Compositions and methods for regulated protein expression in gut

ABSTRACT

The invention provides compositions and methods useful for treating disorders treatable by producing a protein in a regulatable manner in a mucosal cell or tissue of an animal. The treatment methods include in vivo and ex vivo methods, including transplanting in vitro transformed cells that secrete the protein into a mammalian subject.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/015,183, filed Jan. 16, 2008, which claims priority toapplication Ser. No. 09/804,409, filed Mar. 12, 2001, which is now U.S.Pat. No. 7,335,646, Issued Feb. 26, 2008, which claims priority toapplication Ser. No. 60/188,796, filed Mar. 13, 2000 and applicationSer. No. 60/254,464, filed Dec. 8, 2000, all of which are hereinincorporated by reference in their entirety.

TECHNICAL FIELD

This invention relates to regulatable production of proteins in the gut,and more particularly to nutrient regulated production ofglucose-lowering factors from gut endocrine cells.

BACKGROUND

Peptides and proteins, by virtue of their conformational versatility andfunctional specificity, have been used in treating a host of diseasesincluding diabetes, hemophilia, cancer, cardiovascular disorders,infectious diseases and arthritis (Russell C. S. & Clarke L. A. ClinGent 55(6):389 (1999); Ryffel B. Biomed environ Sci 10:65 (1997); KothsK. Curr Opin Biotechnol 6:681 (1995); Buckel P. Trends Pharmacol Sci17:450 (1996)). Presently, more than two thirds of the approved biotechmedicines are systemic protein drugs. With recent advances in the fieldof functional genomics, proteomics and genetic engineering, anincreasing number of protein drugs are entering the biopharmaceuticalmarket.

Originally, protein drugs were purified from animal tissues or humanserum. Protein-based pharmaceuticals have gone through several stages ofimprovement to reach the current state of clinical application. Forexample, the biopharmaceutical industry now uses genetically engineeredyeast and bacteria to manufacture recombinant human proteins (Scopes R.K. Biotechnol Appl Biochem 23:197 (1996)). This groundbreakingtechnology has overcome the health risk and shortages that plagued thefirst generation of protein drugs, and has consequently improved thetherapeutic value of proteins. However, despite these advances, broadusage of proteins as therapeutics is still hampered by difficulties inpurifying recombinant proteins in active forms and the high cost ofmanufacturing procedures (Berthold W. & Walter J. Biologicals 22:135(1994); Scopes R. K. Biotechnol Appl Biochem 23:197 (1996)).Additionally, protein drugs face barriers to their entry into the body.When taken orally, they are susceptible to break down by enzymes in thegastrointestinal tract (Wang W. J Drug Target 4:195 (1996); Woodley J.F. Crit. Rev Ther Drug Carrier Syst 11:61 (1994)).

Other routes of protein delivery explored include infusion pumps (Bremeret. al., Pharm Biotechnol 10:239 (1997)) transdermal delivery (BurkothT. L. Crit. Rev Ther Drug Carrier Syst 16:331 (1999)),microencapsulation (Cleland J. L. Pharma Biotechnol 10:1 (1997)) andinhalation (Gonda I. J Pharm Sci 89:940 (2000)). Currently, subcutaneousand intravenous administration by needle injection is the route ofchoice for delivering protein therapeutics. Unfortunately, this mode ofdelivery is less than ideal because protein concentrations often are notmaintained within a therapeutic range or provide appropriate deliverykinetics. Furthermore, effective treatment with protein drugs usuallyrequires frequent needle injections that can cause local reactions anddiscomfort, hence resulting in poor patient compliance (Jorgensen J. T.J Pediatr Endocrinol 7:175 (1994)). These and other factors limit thetherapeutic application of many drugs and ultimately hinder theircommercial potential. Therefore, it is axiomatic to identify newdelivery methods for protein therapeutics.

Insertion of genes encoding specific therapeutic proteins into cells ofthe body has been used to solve the aforementioned delivery problems intreating diseases. This methodology is referred to as gene therapy andit promises to be the new direction in protein delivery. By thisapproach, cells in the body can be transformed into ‘bioreactors’,manufacturing sufficient quantities of therapeutic proteins and henceeliminating the need for frequent needle injections. Currently, genetherapy can be categorized into two general approaches (Drew J. & MartinL-A. In: Lemoine N. R. (ed) Understanding Gene Therapy. Springer-Verlag,New York, Chp. 1: pp 1-10 (1999)).

In the first approach, referred to as in vivo gene therapy, a gene isintroduced in a form that allows its absorption by cells located withinthe living host. For example, a therapeutic gene is packaged into thegenome of viruses such as retrovirus, adeno-associated virus oradenovirus. The recombinant virus containing the therapeutic gene isthen introduced into a living organism and allowed to infect cellswithin the organism. Through the infection process, the virusincorporates its genome containing the therapeutic genes into thegenomic structure of the host cell. As a result, the infected cellexpresses the therapeutic gene.

The second approach involves in vitro transfer of genetic material tocells removed from the host organism. Following successful incorporationof a gene into the cell's genome, the transformed cells are implantedback into the host. This gene transfer method is referred to as ex vivogene therapy.

Both in vivo and ex vivo gene therapy offer physicians the power to addor modify specific genes resulting in disease cure (Friedmann T. In:Friedmann T (ed) The Development of Human Gene Therapy. Cold SpringHarbor Laboratory Press, Cold Spring Harbor. Chp 1:pp 1-20 (1999)).Clinical applications of this technology are being studied in a widerange of diseases, including cancer, cardiovascular disorders, metabolicdiseases, neurodegenerative disorders, immune disorders and othergenetic or acquired diseases ((Friedmann T. In: Friedmann T (ed) TheDevelopment of Human Gene Therapy. Cold Spring Harbor Laboratory Press,Cold Spring Harbor. Chp 1:pp 1-20 (1999); Drew J. & Martin L-A. In:Lemoine N. R. (ed) Understanding Gene Therapy. Springer-Verlag, NewYork, Chp. 1: pp 1-10 (1999)). Sustained therapeutic concentrations ofnumerous proteins have been achieved after stable introduction of genesthat encode the proteins into cells by gene therapy methodologies.However, for some disorders, regulated delivery of the therapeuticprotein is required. For example, insulin replacement therapy fordiabetic patients ideally requires that the appropriate amount ofinsulin be delivered during meals. Likewise, optimal effectiveness ofappetite suppressants may be achieved via meal-dependent release.Therefore, to deliver such therapeutic proteins, a release systemtriggered by a signal or stimuli, such as a meal, is optimal.

A particular disease well suited for timed delivery is diabetesmellitus, a debilitating metabolic disease caused by absent (type 1) orinsufficient (type 2) insulin production from pancreatic β-cells (Unger,R. H. et al., Williams Textbook of Endocrinology Saunders, Philadelphia(1998)). β-cells are specialized endocrine cells that manufacture andstore insulin for release following a meal (Rhodes, et. al. J. CellBiol. 105:145 (1987)) and insulin is a hormone that facilitates thetransfer of glucose from the blood into tissues where it is needed.Patients with diabetes must frequently monitor blood glucose levels andmany require multiple daily insulin injections to survive. However, suchpatients rarely attain ideal glucose levels by insulin injection(Turner, R. C. et al. JAMA 281:2005 (1999)). Furthermore, prolongedelevation of insulin levels can result in detrimental side effects suchas hypoglycemic shock and desensitization of the body's response toinsulin. Consequently, diabetic patients still develop long-termcomplications, such as cardiovascular diseases, kidney disease,blindness, nerve damage and wound healing disorders (UK ProspectiveDiabetes Study (UKPDS) Group, Lancet 352, 837 (1998)).

Gene therapy represents a promising means to achieve physiologicdelivery of therapeutic peptides such as insulin for the treatment ofdiabetes (Leibowitz, G. & Levine, F. Diabetes Rev. 7:124 (1999)).Surrogate cells that express the incorporated gene, process and storethe encoded protein, and secrete insulin in regulated fashion thereforeaffords a treatment for diabetes. Controlling plasma insulin levels bycoupling insulin production to changing nutrient requirements of thebody also reduces the side effects associated with insulin injection.Accordingly, there is a need for controlled release of proteins toachieve effective treatment of diabetes and other diseases in humans.The present invention satisfies this need and provides relatedadvantages.

SUMMARY

The present invention is based, in part, on the production oftransformed gut cells that produce insulin in response to glucose.Transformed glucose-responsive cells present in the gut of animals areable to secrete insulin at physiological levels that restore normalglucose homeostasis in diabetic animals. Thus, gut endocrine cells aresuitable targets for therapeutic introduction of nucleic acid encodingproteins, ex vivo or in vivo, whose production in an animal in responseto a signal or stimuli (e.g., a nutrient) provides a therapeuticbenefit.

The invention therefore provides methods of generating a mucosal cellthat produces a protein in response to a nutrient, and compositionsincluding a mucosal cell that produces a protein in response to anutrient. In one embodiment, a method includes contacting a mucosal cellwith a polynucleotide comprising an expression control element inoperable linkage with a nucleic acid encoding a protein under conditionsallowing transformation of the cell; and identifying a cell transformantthat produces the protein in a nutrient-regulatable manner, therebygenerating a mucosal cell that produces a protein in response to anutrient. In another embodiment, a composition includes an isolated orcultured mucosal cell that produces a protein regulatable by a nutrient,wherein expression of the protein is conferred by a transgene comprisingan expression control element in operable linkage with a nucleic acidencoding the protein.

The invention therefore also provides methods of treating a subjecthaving or at risk of having a disorder treatable by producing a proteinin a tissue. In one embodiment, a method includes implanting one or moremucosal cells that produce a protein in response to a nutrient into thetissue in an amount effective for treating the disorder. Exemplaryimplantable tissues include mucosal (e.g., gastrointestinal tract) andnon-mucosal (e.g., liver, pancreas or muscle) tissues.

Mucosal cells included in the invention include cells that respond tonutrient, which increases expression (e.g., via a nutrient-regulatableexpression control element) or secretion of the protein (e.g., secrete asynthesized protein in response to a signal or stimuli, i.e., a“secretagogue”).

Nutrients included are natural and non-natural ingestible compounds,such as a sugar, fat, carbohydrate or starch, an amino acid orpolypeptide, a triglyceride, a vitamin, a mineral, or cellulose.Nutrient-regulatable elements include a gut endocrine promoter, such asa glucose-dependent insulinotropic polypeptide (GIP) promoter.Nutrient-regulatable elements include functional variants thereof (e.g.,point mutation) or a functional subsequence of a full-length regulatableelement (deleted sequence). Expression control elements in operablelinkage with a nucleic acid encoding the protein, can further include avector (e.g., a viral vector).

Mucosal cells included in the invention are obtained from a subject,such as a mammal (e.g., human), are obtained from a tissue or organ ofthe gastrointestinal tract or are derived from a cultured cell line ofgut origin. Exemplary tissues where mucosal cells can be obtainedinclude the gastrointestinal tract, large or small intestine (jejunum,duodenum), stomach, esophagus, buccal or mouth tissue. Mucosal cellsalso include those that can or are adapted for growth in mucosum, evenfor short periods of time. Mucosal cells include endocrine andnon-endocrine cells, K-cells, stem cells, L-cells, S-cells, G-cells,D-cells, I-cells, Mo-cells, Gr-cells and entero-endocrine cells.

Invention compositions and methods include therapeutic proteins such asinsulin, leptin, GLP-1, GLP-2, cholecystokinin, a glucagon antagonist,Ghrelin, growth hormones, clotting factors, or antibodies.

The invention therefore also provides methods of treating a subjecthaving, or at risk of having, a disorder treatable by producing atherapeutic protein in a mucosal tissue. In one embodiment, a methodincludes contacting mucosal cells in the subject that have beentransformed with a polynucleotide, for example, an expression controlelement in operable linkage with a nucleic acid encoding the therapeuticprotein, with a nutrient that induces production of the protein in anamount effective to treat the disorder.

Conditions and disorders treatable with the invention methods andcompositions include hyperglycemic conditions, such as insulin-dependentand -independent diabetes or where fasting plasma glucose levels aregreater than 110 mg/dl; obesity or an undesirable body mass.

The invention therefore also provides animal and genetic models. In oneembodiment, a non-human transgenic animal that produces a therapeuticprotein (e.g., insulin) in a mucosal tissue is provided. In one aspect,therapeutic protein production does not naturally occur in the mucosaltissue of the animal, is conferred by a transgene present in the mucosaltissue, and the transgene includes a polynucleotide including anexpression control element in operable linkage with a nucleic acidencoding the protein, wherein production of the protein in the mucosaltissue of the animal is responsive to the nutrient. In one aspect, theprotein comprises insulin. The transgenic animal can therefore be maderesistant to developing a hyperglycemic condition. A transgenic animalhaving or at risk of having a hyperglycemic condition can therefore bemade to have less glucose or less likely to develop hyperglycemia. Inanother aspect, the animal is a mouse (e.g., diabetic or hyperglycemicor obese mouse). In yet another aspect, the expression control elementconferring expression comprises a nutrient-regulatable element, afunctional variant thereof, or a functional subsequence thereof. Instill another aspect, the expression control element includes aglucose-inducible promoter, for example, a glucose-dependentinsulinotropic polypeptide (GIP) promoter. Expression of the protein inthe animal can be conferred in gastrointestinal tract, intestine/gut,stomach. Cells or tissues of the transgenic animal that produce insulinin response to the nutrient can be isolated. Cells that express proteinand also can be isolated include K cells, stem cells and endocrine ornon-endocrine cells.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows visualization of green fluorescence protein (GFP)expression driven by the GIP promoter in tumor-derived intestinalendocrine cells, STC-1. The left panel shows a sample bright-fieldpopulation of cells. The same field is seen in the right panel underfluorescence allowing identification of GFP-expressing cells.Fluorescent cell clusters were selected and expanded in culture togenerate the K cell line, GTC-1.

FIG. 2 is a representative Northern blot analysis of GIP mRNA in STC-1and GTC-1 cells showing that GTC-1 is a highly enriched population ofGIP-producing K cells.

FIG. 3 is a schematic view of the GIP/Ins plasmid construct used fortargeting human insulin expression to K cells. It contains the genomicsequence of the human insulin gene operably linked to the GIP promoter(˜2.5 kb of 5′-regulatory sequence of the GIP gene The three exons aredenoted by filled boxes (E1, 2 and 3). The positions of primers used forRT-PCR detection of proinsulin mRNA are indicated. Hind III (H), Pvu II(P) and Xho I (X) sites are shown. Positions of start (ATG) and stopcodons are indicated.

FIG. 4 shows expression of human insulin and proinsulin processingenzymes in tumor-derived K cells. The upper panel shows RT-PCR analysisof cDNA from human islets (H) and GTC-1 cells either transfected (T) oruntransfected (UT) with GIP/Ins plasmid. Samples were prepared with (+)or without (−) reverse transcriptase. The lower panel shows immunoblotanalysis of proprotein convertases PC1/3 and PC2 expression in GTC-1cells and a β-cell line (INS-1). Arrows indicate predicted product sizefor PC1/3 isoforms (64 and 82 kD) and PC2 isoforms (66 and 75 kD).

FIG. 5 shows the levels of human insulin and C-peptide detected inculture media from GTC-1 cells transfected (T) or untransfected (UT)with the GIP/Ins construct. Insulin and C-peptide are indicated by openand solid bars respectively.

FIG. 6 is a graph showing the stimulatory effect of glucose on insulinsecretion from GTC-1 cells stably transfected with the GIP/Ins plasmid.

FIG. 7 shows co-expression of glucokinase (GK, red) and GIP (green) inmouse duodenal sections.

FIG. 8 shows genomic Southern blot and PCR identification of transgenicfounder lines. Mouse numbers are indicated at the top.

FIG. 9 shows targeted expression of human insulin to K cells intransgenic mice harboring the GIP/Ins construct. The upper panel shows arepresentative Northern blot analysis for human insulin gene expressionin human islets, control duodenum (mouse) and transgenic mouse tissues.The lower panel shows RT-PCR analysis of cDNA from human islets (H),mouse islets (M) and duodenum samples (D) from two transgenic mice usinghuman or mouse specific proinsulin primers. Samples were prepared in thepresence (+) or absence (−) of reverse transcriptase. Ø indicates no DNAand M indicates markers.

FIG. 10 shows the results of immunohistochemical staining for humaninsulin in sections of stomach (left panel) and duodenum (middle panel)from a transgenic mouse. Arrows indicate human insulin immunoreactivecells. The right panel shows duodenal sections from the same animalexamined by immunofluorescence microscopy following co-staining withantisera specific for insulin (INS, green) and GIP (red).

FIG. 11A is a graph showing that the production of human insulin fromgut K cells of transgenic mice is meal-regulated.

FIG. 11B is a graph showing the release kinetics of human insulin fromgut K cells of transgenic mice in response to a mixed meal test and oralglucose challenge.

FIG. 12 is a graph showing the changes of blood glucose concentration innormal control mice, streptozotocin (STZ)-treated control mice andSTZ-treated transgenic mice following an oral glucose challenge (1.5 gglucose/kg body weight).

FIG. 13 is a series of micrographs showing immunohistochemical stainingfor mouse insulin in pancreatic sections from control and STZ-treatedtransgenic mice. Arrows indicate islets.

FIG. 14 are nucleotide sequences of rat GIP and mouse chromagranin Agene promoter regions.

FIG. 15 are nucleotide sequences of promoter and exon 1 of mousesecretogranin II (Accession no. AF037451) and a 5′ portion of mouseglucokinase gene promoter (Accession no. U93275).

FIG. 16 are nucleotide sequences of a 3′ portion of mouse glucokinasegene promoter (Accession no. U93275), human adenosine deaminase genepromoter region (Accession no. X02189); and human pre-proinsulin aminoadic sequence, and 60 bp of a 5′ region of pre-proinsulin.

FIG. 17 are nucleotide sequences of the remaining 3′ portion of humanpre-proinsulin and a 5′ portion of the human leptin gene cDNA.

FIG. 18 are nucleotide sequences of the remaining 3′ portion of humanleptin, human CCK amino acid and nucleotide sequences and 60 bp of ratCCK promoter.

FIG. 19 are nucleotide sequences of the remaining 3′ portion of rat CCKpromoter and amino acid and nucleotide sequences of human growthhormone.

FIG. 20 is the sequence for the rat GIP promoter from −1 to −1894 bp.

DETAILED DESCRIPTION

The invention is based, in part, on the targeted production of a proteinin a tissue of animals at levels sufficient to provide therapy. Morespecifically, the invention includes methods of targeting expression ofany protein of interest to endocrine cells in the gastrointestinal tractof a subject such that the protein is released into the bloodstream ofthe subject in a regulated manner. Genetic constructs including anexpression control element (e.g., promoter) that targets gene expressionto gut endocrine cells operably linked to nucleic acid encoding atherapeutic protein can be used. When the gene construct is incorporatedinto the endocrine cells, the encoded protein will be expressed andsecreted in a regulated manner. The transformed endocrine cellsexpressing the protein encoded by the nucleic acid of interest cansecrete a therapeutically effective amount of the protein into thebloodstream of the subject upon feeding of a substance (e.g., nutrient)that increases production of the protein.

Delivery of a genetic construct comprised of a GIP promoter operablylinked to a human insulin gene in mice successfully targeted expressionand secretion of human insulin by K cells in the gastrointestinal tractof transgenic offspring. Furthermore, the production of human insulin inthe transgenic animals was meal regulated. The amount of insulinsecreted by the cells was sufficient to protect the transgenic mice fromdeveloping diabetes after destruction of pancreatic β-cells. Insulinproduction was also sufficient to provide normal glucose homeostasis.Thus, introduction of a gene encoding therapeutic proteins such asinsulin into meal-regulated endocrine cells in the gut of an animal,either by in vivo or by ex vivo methods (e.g., transplanting in vitrotransformed cells that secrete insulin into an animal), can be used totreat disorders treatable by production of a protein.

In accordance with the invention, there are provided methods ofgenerating a mucosal cell that produces a protein regulatable by anutrient. A method of the invention includes contacting a mucosal cellwith a polynucleotide comprising an expression control element inoperable linkage with a nucleic acid encoding a protein under conditionsallowing transformation, and identifying a transformed cell thatproduces the protein in a nutrient-regulatable manner. In oneembodiment, the mucosal cell is contacted with the polynucleotide invivo. In another embodiment, the mucosal cell is contacted with thepolynucleotide in vitro. In yet another embodiment, the mucosal cellcontacted with the polynucleotide in vitro is suitable fortransplantation into an animal. In additional embodiments, the mucosalcell is an endocrine cell (e.g., a K cell), or a non-endocrine cell. Instill further embodiments, the mucosal cell is a stem cell or apluripotent or multipotent progenitor cell.

In another embodiment, a nucleic acid expression construct used in theinvention is designed to target production of proteins ingastrointestinal endocrine cells. The construct contains an expressioncontrol element operably linked to desired nucleic acid sequences.Expression control elements include promoters capable of targetingexpression of a linked nucleic acid of interest to endocrine cells inthe gut. Introduction of constructs into target cells can be carried outby conventional methods well known in the art (osmotic shock (e.g.,calcium phosphate), electroporation, viral vectors, vesicles or lipidcarriers (e.g., lipofection), direct microinjection, etc.).

Typically cell transformation employs a vector. The term “vector,”refers to, e.g., a plasmid, virus, such as a viral vector, or othervehicle known in the art that can be manipulated by insertion orincorporation of a polynucleotide, for genetic manipulation (i.e.,“cloning vectors”), or can be used to transcribe or translate theinserted polynucleotide (i.e., “expression vectors”). Such vectors areuseful for introducing polynucleotides, including a nutrient-regulatableexpression control element in operable linkage with a nucleic acid, andexpressing the transcribed antisense or encoded protein in cells invitro or in vivo.

A vector generally contains at least an origin of replication forpropagation in a cell. Control elements, including expression controlelements (e.g., nutrient-regulatable) as set forth herein, presentwithin a vector, are included to facilitate transcription andtranslation. The term “control element” is intended to include, at aminimum, one or more components whose presence can influence expression,and can include components other than or in addition to promoters orenhancers, for example, leader sequences and fusion partner sequences,internal ribosome binding sites (IRES) elements for the creation ofmultigene, or polycistronic, messages, splicing signal for introns,maintenance of the correct reading frame of the gene to permit in-frametranslation of mRNA, polyadenylation signal to provide properpolyadenylation of the transcript of a gene of interest, stop codons,among others.

Vectors can include a selection marker. As is known in the art,“selection marker” or equivalents means genes that allow the selectionof cells containing the gene. “Positive selection” refers to a processwhereby only the cells that contain the positive selection marker willsurvive upon exposure to the positive selection agent or be marked. Forexample, drug resistance is a common positive selection marker; cellscontaining the positive selection marker will survive in culture mediumcontaining the selection drug, and those which do not contain theresistance gene will die.

Suitable drug resistance genes are neo, which confers resistance toG418, or hygr, which confers resistance to hygromycin, or puro whichconfers resistance to puromycin, among others. Other positive selectionmarker genes include genes that allow the sorting or screening of cells.These genes include genes for fluorescent proteins, the lacZ gene, thealkaline phosphatase gene, and surface markers such CD8, among others.

Vectors included in the invention can contain negative selectionmarkers. “Negative selection” refers to a process whereby cellscontaining a negative selection marker are killed upon exposure to anappropriate negative selection agent which kills cells containing thenegative selection marker. For example, cells which contain the herpessimplex virus-thymidine kinase (HSV-tk) gene are sensitive to the druggancyclovir (GANC). Similarly, the gpt gene renders cells sensitive to6-thioxanthine.

Vectors included in the are those based on viral vectors, such as simianvirus 40 (SV40) or bovine papilloma virus (BPV), which has the abilityto replicate as extra-chromosomal elements (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982; Sarver et al., Mol.Cell. Biol. 1:486 (1981)). Viral vectors include retroviral,adeno-associated virus, adenovirus, reovirus, lentivirus, rotavirusgenomes etc, modified for introducing and directing expression of apolynucleotide or transgene in mucosal cells (Cone et al., Proc. Natl.Acad. Sci. USA 81:6349 (1984)).

“Expression control elements” include polynucleotides, such as promotersand enhancers, that influence expression of an operably linked nucleicacid. Expression control elements and promoters include those active ina particular tissue or cell type, referred to herein as a“tissue-specific expression control elements/promoters.” Tissue-specificexpression control elements are typically active in specific cell ortissue because they are recognized by transcriptional activatorproteins, or other regulators of transcription, that are unique to aspecific cell or tissue type.

A particular class of a tissue specific promoter is a “gut endocrinecell specific promoter,” a promoter that drives expression of anoperably linked nucleic acid in a gut endocrine cell. The GIP promoteris a specific example of a gut endocrine cell promoter. The GIP promoterincludes multiple regulatory sequences which confer specific expressionin the gastrointestinal tract (Tseng, C. C. et al. Proc. Natl. Acad.Sci. USA 90:1992 (1993); Yeng, C. M., et al. Mol. Cell. Endocrinol.154:161 (1999)). A GIP promoter sized about 2.5 Kb (−1 to ˜−2500 bp) inlength targeted transgene expression to the stomach and duodenum (FIGS.9 & 10). A shorter GIP promoter (−1 to ˜−1200 bp) conferred expressionof transgene in the stomach but not duodenum and miss-targeted transgeneexpression to the pancreas (Yeng, C. M., et al. Mol. Cell. Endocrinol.154:161 (1999)). The regulatory sequence between −1200 to −2500 bptherefore appears necessary for targeting transgene expression to GIPproducing cells in the intestine.

Characterization of transcriptional elements in the GIP promoterrevealed two TATA boxes (−27 to −23 and −115 to −111) and two CCAAT-likeboxes (−158 to −154 and −170 to −167), potential AP-1 and AP-2 sites,cAMP response element (CRE), and a potential insulin response element(IRE) upstream of the putative transcription start site. Two putativeGATA binding motifs also have been identified in the GIP promoter (−178to −172 (proximal GATA); CAGATAC and −190 to −184 (distal GATA);CAGATAA) which conform to the consensus GATA binding motif sequence,(A/T)GATA(A/G). Specific mutations in the GIP promoter distal andproximal GATA motifs resulted in approximately 90% and 35% reduction inGIP promoter activity respectively, as assessed by luciferase reporterexpression (Boylan et al., J. Biol. Chem. 273:17438 (1997)). However, aGIP promoter with both GATA motifs mutated behaved the same as thepromoter with only the distal GATA motif altered. Thus, a GIP promotercontaining one or more of the aforementioned nucleotide sequences orvariants is an example of a subsequence that can retainglucose-regulatable or tissue specific (gut) expression of an operablylinked nucleic acid. Such subsequences and variants can be used toconfer glucose-regulatable or cell specific expression of an operablylinked nucleic acid in vitro or in vivo.

An additional example of a tissue-specific control element is thepromoter of the proglucagon gene. Similar to the GIP promoter, theproglucagon promoter has multiple control sequences that conferexpression in either the gastrointestinal tract or brain and pancreas(Lee, Y. C., et al. J. Biol. Chem. 267:10705 (1992); Gajic and Drucker,Endocrinol. 132:1055 (1993). A1300 bp portion of the upstream ratproglucagon promoter sequence targeted expression of a transgene in thebrain and pancreas, but not in the gastrointestinal tract (Efrat S., et.al. Neuron 1:605 (1988)). A longer proglucagon gene promoter (−1 to˜−2000 bp) directed transgene expression in the intestine, in additionto brain and pancreas (Lee, Y. C., et al. J. Biol. Chem. 267:10705(1992)). Thus, the portion conferring expression in gut appears to bewithin a 700 bp region of the promoter located between 1300 and 2000 bpupstream of the proglucagon gene.

Additional tissue-specific expression control elements that may beemployed to target the expression of the nucleic acid of interest in gutendocrine cells are listed in Table 1. Many of these promoters are alsonutrient-regulatable elements. For example, the GIP promoter includesmultiple regulatory sequence which confer expression of an operablylinked nucleic acid in response to nutrients.

This list is not intended to be exhaustive of all the possibleexpression control elements useful for driving gene expression in gutendocrine cells but merely to be exemplary.

Although tissue-specific expression control elements may be active inother tissue, for example, a gut specific expression control element maybe active in a non-gut tissue, expression is significantly less thanthat in the gut tissue, (e.g., for non-gut tissue 6-10 fold less than ina gut tissue). Targeted delivery of a vector to gut tissue can limit thepossibility of expression elsewhere in the body (e.g., in non-targettissues). Accordingly, tissue-specific elements included herein need nothave absolute tissue specificity of expression.

TABLE 1 Exemplary Promoters and Enhancers for Targeting Expression ofProteins to Endocrine Cells in the Gut Glucokinase Chromogranin A and BCholecystokinin Glucose-dependent insulinotropic polypeptide ProglucagonAdenosine deaminase Secretin Gastrin Somatostatin Motilin Ghrelin

Additional expression control elements can confer expression in a mannerthat is regulatable, that is, a signal or stimuli increases or decreasesexpression of the operably linked nucleic acid. A regulatable elementthat increases expression of the operably linked nucleic acid inresponse to a signal or stimuli is also referred to as an “inducibleelement” (i.e., is induced by a signal, e.g., a nutrient). A regulatableelement that decreases expression of the operably linked nucleic acid inresponse to a signal or stimuli is referred to as a “repressibleelement” (i.e., the signal decreases expression such that when thesignal, is removed or absent, expression is increased). Typically, theamount of increase or decrease conferred by such elements isproportional to the amount of signal or stimuli present; the greater theamount of signal or stimuli, the greater the increase or decrease inexpression.

A particular example of a regulatable expression control element is anelement that increases or decreases expression of an operably linkednucleic acid in response to or withdrawal of a nutrient, in which casethe element is referred to as a “nutrient-regulatable element.” Anutrient inducible or repressible element generally provides basallevels of transcription (i.e., levels of expression in the absence of astimuli or signal). Typically, basal levels of transcription are greaterfor a repressible element than for an inducible element.

As used herein, the term “nutrient” means any ingestible or consumablematerial such as that present in food or drink. As there are many,perhaps billions of different organic and inorganic substances presentin food or drink, the term is used broadly herein. Particular examplesof nutrients include sugars (e.g., glucose, lactose, sucrose, fructose,mannose, etc.), carbohydrates, starches, fats (saturated orunsaturated), lipids, fatty acids, triglycerides, polypeptides, aminoacids, cellulose, hormones, vitamins, and minerals.

Nutrients may also modulate translation or stability of a protein.“Nutrient-regulatable” therefore includes situations where the nutrientmodulates transcription, translation of the transcript into protein, orstability of the protein, thereby increasing or decreasing the amount oftranscript or protein.

An expression control element can be “constitutive,” such thattranscription of the operably linked nucleic acid occurs without thepresence of a signal or stimuli. Additionally, expression controlelements also include elements that confer expression at a particularstage of the cell cycle or differentiation. Accordingly, the inventionfurther includes expression control elements that confer constitutive,regulatable (e.g., nutrient-regulatable), tissue-specific, cell cyclespecific, and differentiation stage specific expression.

Expression control elements include full-length sequences, such asnative promoter and enhancer elements, as well as subsequences orpolynucleotide variants which retain all or part of full-length ornon-variant function (e.g., retain some amount of nutrient regulation orcell-specific expression). As used herein, the term “functional” andgrammatical variants thereof, when used in reference to a nucleic acidor polypeptide sequence, subsequence or fragment, or nucleotide or aminoacid sequence variant, means that the sequence has one or more functionsof native nucleic acid or polypeptide sequence (e.g., non-variant orunmodified sequence). As used herein, the term “variant” means asequence (nucleotide or amino acid) substitution (e.g., point mutation),deletion (internal or external) or addition (e.g., chimericpolypeptide), or other point mutation modification (e.g., chemicalderivatives such as modified forms resistant to proteases or nucleases).Typically, amino acid variants have a few or several amino acid changes(e.g., 1 to 10, 10 to 20, 20 to 50) such as one or more conservativeamino acid substitutions, or non-conservative amino acid substitutionsoutside of domains critical to a functionality that is desired to beretained in the variant (e.g., for insulin, glucose lowering function).Expression control elements, such as nutrient-regulatable elements, alsoinclude functional variants, or subsequences. For example, a subsequenceof a glucose-regulatable ˜2.5 Kb GIP promoter (e.g., 2 Kb, 1 Kb, 0.5 Kb,0.25 Kb, 0.20 Kb, 100 bp or less) can retain glucose-regulatable ortissue specific (gut or pancreas or brain) expression of an operablylinked nucleic acid. Functional domains of various promoters havingknown properties can be configured to optimize amounts and patterns ofexpression of the operably linked nucleic acid.

Expression control elements included herein can be from bacteria, yeast,plant, or animal (mammalian or non-mammalian), so long as they functionto confer expression control of an operably linked nucleic acid. Thus,any expression control element induced by a substance or stimuli (e.g.,nutrient) from any organism can be used to modulate transcription of anoperably linked nucleic acid in a mucosal cell and, as appropriate,translation of the encoded protein in response to the substance orstimuli, as set forth herein.

Nutrient-regulatable expression control elements exist, for example, aspromoters that regulate expression of enzymes involved in glycolysis,lipid metabolism, carbohydrate metabolism and cholesterol (e.g.,steroid) metabolism, which are modulated by sugars, fats, carbohydrate,and cholesterol, respectively, and are applicable in the invention.Particular examples of nutrient-regulatable control elements are glucoseinducible elements that drive expression of L-pyruvate kinase,acetyl-CoA-carboxylase, spot-14, fatty acid synthase, glyceraldehydephosphate dehydrogenase phospho-enol-pyruvate carboxykinase,glucose-6-phosphatase and phosphofructokinase (see, also, e.g., Rutter,G A et al., News Physiol Sci. 15:149 (2000)). Another example of anutrient-regulatable control element is the alcohol-dehydrogenase generegulatory element. Yet another example of a nutrient-regulatablecontrol element is the vitamin-D response element, which confersexpression in the presence of vitamin D. The mammalian metallothioneingene promoter is an expression control element inducible by metals. Aswith tissue-specific control elements, nutrient-regulatable controlelements may be responsive to multiple nutrients. For example, aglucose-inducible element may also be responsive to lactose. Aparticular nutrient (e.g., glucose) is therefore not meant to beexclusive of other nutrients in that other nutrients may modulateactivity (increase or decrease), to a lesser degree, of the controlelement.

An example of a bacterial nutrient-regulatable expression controlelement is the lac repressor, which is inducible by beta-galactosides.An example of a yeast nutrient-regulatable expression control element isthe gal promoter present in GAL1 and GAL10 genes, which confergalactose-inducible expression. These elements can be operably linked toa nucleic acid and introduced into a mucosal cell in order to confernutrient-regulatable production of the encoded protein.

Additional expression control elements included are those that areresponsive to non-nutrients. Particular examples are chemicals or drugsthat are orally active but not normally found in food. The non-nutrientdrug or chemical, when consumed, stimulates expression of a nucleic acidoperably linked to the non-nutrient expression control element.Ingesting specific amounts of the chemical or drug provides control ofthe amount of nucleic acid or protein produced (via transcription orsecretion). For example, where a drug inducible expression controlelement confers expression of a nucleic acid encoding insulin, greateramounts of insulin can be produced in the gut by increasing the amountof drug consumed. Particular examples of such non-nutrient expressioncontrol systems can be found, for example, in U.S. Pat. Nos. 5,989,910;5,935,934; 6,015,709; and 6,004,941.

As used herein, the term “operable linkage” or grammatical variationsthereof refers to a physical or functional juxtaposition of thecomponents so described as to permit them to function in their intendedmanner. In the example of an expression control element in operablelinkage with a nucleic acid, the relationship is such that the controlelement modulates expression of the nucleic acid.

Expression control can be effected at the level of transcription,translation, splicing, message stability, etc. Typically, an expressioncontrol element that modulates transcription is juxtaposed near the 5′end of the transcribed nucleic acid (i.e., “upstream”). Expressioncontrol elements can also be located at the 3′ end of the transcribedsequence (i.e., “downstream”) or within the transcript (e.g., in anintron). Expression control elements can be located at a distance awayfrom the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to5000, or more nucleotides from the nucleic acid). Expression of theoperably linked nucleic acid is at least in part controllable by theelement (e.g., promoter) such that the element modulates transcriptionof the nucleic acid and, as appropriate, translation of the transcript.A specific example of an expression control element is a promoter, whichis usually located 5′ of the transcribed sequence. Another example of anexpression control element is an enhancer, which can be located 5′, 3′of the transcribed sequence, or within the transcribed sequence.

As used herein, the term “produces” or “production,” when used inreference to a protein expressed by a mucosal cell or tissue, meanseither expression or secretion of the protein by a mucosal cell. Thus,where a mucosal cell produces a protein in response to a signal orstimuli, such as a nutrient, expression or secretion of the proteinincreases over the amount prior to the signal or stimuli. Production ofa protein by the mucosal cell or tissue may be due to increasedtranscription of the nucleic acid, translation of the transcript,stability of the transcript or protein, or secretion of the encodedprotein. Typically, secretion of a protein by a cell increased by asignal or stimuli (i.e. a secretagogue) stimulates release of a proteinalready translated in the cell. Proteins whose secretion is regulatedare typically stored in secretory vesicles within endocrine cells.

Alternatively, transcription or translation of a nucleic acid encodingthe protein, and subsequent secretion of the translated protein, may beincreased by a signal or stimuli. Thus, in the example of anon-endocrine cell, a signal or stimuli (e.g., nutrient) may stimulatetranscription of nucleic acid encoding the protein (e.g., insulin) via anutrient-inducible expression control element, and the cell willsubsequently secrete the encoded protein following its translation. Inthe example of an endocrine cell, such as a gut endocrine cell (e.g.,K-cell, L-cell, etc.), the expression control element used to conferexpression of the protein may or may not be regulatable but in eithercase a signal or stimuli typically will regulate secretion of theprotein from the cell. In this case, the signal or stimuli functions asa secretagogue that stimulates or increases secretion of a protein.Therefore, in endocrine cells, whether expression is or is not nutrientregulatable (e.g., a constitutive promoter), protein production by thecell is nutrient regulatable because secretion of the protein ismodulated by the nutrient. Accordingly, “nutrient-regulatable” alsorefers to nutrient modulating secretion of a protein from a cell.

Increased secretion of a protein by an endocrine cell in response to asignal or stimuli provides a more rapid response to the signal orstimuli in comparison to a protein produced by increasing transcriptionof a nucleic acid encoding the protein and subsequent secretion, as in anon-endocrine cell. In contrast, in a non-endocrine cell, a signal orstimuli such as a nutrient can increase transcription of a nucleic acidencoding a protein, and the translated protein is subsequently secretedby the cell without need for a signal or stimuli (e.g., nutrient). Thus,for a non-endocrine mucosal cell transformed with a nutrient-regulatabletransgene, transcription of the transgene will be nutrient inducible,but secretion does not require the nutrient.

The nucleic acid can encode a therapeutic polypeptide, such as insulin,a glucagon antagonist, leptin, GLP-1 or cholecytoskinin. For example, asubsequence of full-length insulin that retains some ability to lowerglucose, provide normal glucose homeostasis, or reduce thehistopatholgical conditions associated with chronic or acutehyperglycemia in vivo is but one example of a functional subsequencethat has one or more activities of its full length counterpart.Similarly, a subsequence or variant of leptin or CCK or a growthhormone, clotting factor or antibody that retains all or some of theability to suppress appetite or induce weight stabilization or weightloss, stimulate growth, decrease clotting time or bleeding episodes, orprovide passive protection against a foreign antigen (e.g., H. pylori)are additional examples of a functional sequence or variant that can beexpressed in mucosal tissue of an animal to provide therapeutic benefit.

Thus, “polypeptides,” “proteins” and “peptides” encoded by the “nucleicacids,” include full-length native sequences, as with naturallyoccurring proteins, as well as functional subsequences, modified formsor sequence variants so long as the subsequence, modified form orvariant retains some degree of functionality of the native full-lengthprotein.

As used herein, the term “transgene” means a polynucleotide that hasbeen introduced into a cell or organism by artifice. For example, amucosal cell having a transgene, the transgene has been introduced bygenetic manipulation or “transformation” of the cell. A cell or progenythereof into which the transgene has been introduced is referred to as a“transformed cell” or “transformant.” Typically, the transgene isincluded in progeny of the transformant or becomes a part of theorganism that develops from the cell. Transgenes may be inserted intothe chromosomal DNA or maintained as a self-replicating plasmid, YAC,minichromosome, or the like.

Transgenes include any gene that is transcribed into an antisense orencodes a polypeptide. Particular polypeptides encoded by transgenesinclude detectable proteins, such as luciferase, β-galactosidase, greenfluorescent protein (for non-invasive in vivo detection),chlorampenicolacetyltransferase, or proteins that are detectable (e.g.,immunologically detectable). Detectable proteins are useful forassessing efficiency of cell transformation (e.g., in in vivo genetransfer), cell implantation success, as measured by cell survival orproliferation, for example (e.g., after implanting the transformed cellinto animal mucosa).

Therapeutic proteins include insulin, a particular transgene useful totreat a hyperglycemic condition such as diabetes. Insulin is the primaryhormonal modulator of glucose metabolism and facilitates transport ofglucose from the blood to key metabolic organs such as muscle, liver andfat. As shown in Example III, insulin production in the gut oftransgenic mice by an insulin transgene prevents diabetes in the mice.Insulin is produced in amounts sufficient to restore glucose toleranceand the timing of insulin release restores normal glucose homeostasis.

Another example of a transgene encoding a therapeutic protein to treat ahyperglycemic condition is a glucagon antagonist. Glucagon is a peptidehormone produced by α-cells in pancreatic islets and is a majorregulator of glucose metabolism (Unger R. H. & Orci L. N. Eng. J. Med.304:1518 (1981); Unger R. H. Diabetes 25:136 (1976)). As with insulin,blood glucose concentration mediates glucagon secretion. However, incontrast to insulin glucagon is secreted in response to a decrease inblood glucose. Therefore, circulating concentrations of glucagon arehighest during periods of fast and lowest during a meal. Glucagon levelsincrease to curtail insulin from promoting glucose storage and stimulateliver to release glucose into the blood. A specific example of aglucagon antagonist is [des-His¹, des-Phe⁶, Glu⁹]glucagon-NH₂. Instreptozotocin diabetic rats, blood glucose levels were lowered by ˜37%within 15 min of an intravenous bolus (0.75 μg/g body weight) of thisglucagon antagonist (Van Tine B. A. et. al. Endocrinology 137:3316(1996)).

Another example of a transgene encoding a therapeutic protein to treat ahyperglycemic condition or undesirable body mass (e.g., obesity) isglucagon-like peptide-1 (GLP-1). GLP-1 is a hormone released fromL-cells in the intestine during a meal which stimulates pancreaticβ-cells to increase insulin secretion. GLP-1 has additional activitieswhich make it an attractive therapeutic agent for treating obesity anddiabetes. For example, GLP-1 reduces gastric emptying, suppressesappetite, reduces glucagon concentration, increases β-cell mass,stimulates insulin biosynthesis and secretion in a glucose-dependentfashion, and likely increases tissue sensitivity to insulin (Kieffer T.J., Habener J. F. Endocrin. Rev. 20:876 (2000)). Therefore, regulatedrelease of GLP-1 in the gut to coincide with a meal can providetherapeutic benefit for a hyperglycemic condition or an undesirable bodymass.

GLP-1 analogs that are resistant to dipeptidyl peptidate IV (DPP IV)provide longer duration of action and improved therapeutic value. Thus,transgenes encoding GLP-1 analogs with increased duration of action canbe tageted to gut using the invention described herein to providenutrient regulated production of GLP-1 analogs for treating ahyperglycemic condition or an undesirable body weight.

Another example of a transgene encoding a therapeutic protein to treat ahyperglycemic condition is an antagonist to the hormone resistin.Resistin is an adipocyte-derived factor for which expression is elevatedin diet-induced and genetic forms of obesity. Neutralization ofcirculating resistin improves blood glucose and insulin action in obesemice. Conversely, administration of resistin in normal mice impairsglucose tolerance and insulin action (Steppan C M et. al. Nature 409:307(2001)). Production of a protein that antagonizes the biological effectsof resistin in gut can therefore provide an effective therapy forobesity-linked insulin resistance and hyperglycemic conditions.

Yet another example of a transgene encoding a therapeutic protein totreat undesirable body mass (e.g., obesity) or a hyperglycemic conditionis leptin. Leptin, although produced primarily by fat cells, is alsoproduced in smaller amounts in a meal-dependent fashion in the stomach.Leptin relays information about fat cell metabolism and body weight tothe appetite centers in the brain where it signals reduced food intake(promotes satiety) and increases the body's energy expenditure. A singledaily subcutaneous injection of leptin had only a modest effect onweight reduction in humans yet leptin treatment results in profounddecreases of fat mass in rodents as well as reduction in blood glucose(Seufert J. et. al. Proc Natl Acad Sci USA. 96:674 (1999). Previousstudies have shown that leptin is rapidly degraded in the circulation.Thus, delivery from gut in a regulated fashion will likely enhance theclinical benefit of leptin reducing food intake and body mass, as wellas blood glucose.

Yet another example of a transgene encoding a therapeutic protein totreat undesirable body weight (e.g. obesity) or a hyperglycemiccondition is the C-terminal globular head domain of adipocytecomplement-related protein (Acrp30). Acrp30 is a protein produced bydifferentiated adipocytes. Administration of a proteolytic cleavageproduct of Acrp30 consisting of the globular head domain to mice leadsto significant weight loss (Fruebis J. et al. Proc. Natl. Acad. Sci. USA98:2005 (2001)). Therefore, targeted expression of a transgene encodingthe globular domain of Acrp30 to gut can promote weight loss.

Still another example of a transgene encoding a therapeutic protein totreat undesirable body mass (e.g., obesity) is cholecystokinin (CCK).CCK is a gastrointestinal peptide secreted from the intestine inresponse to particular nutrients in the gut. CCK release is proportionalto the quantity of food consumed and is believed to signal the brain toterminate a meal (Schwartz M. W. et. al. Nature 404:661-71 (2000)).Consequently, elevated CCK can reduce meal size and promote weight lossor weight stabilization (i.e., prevent or inhibit increases in weightgain). A nutrient-regulated CCK delivery system can therefore providetherapeutic benefit for the purpose of reducing food intake in persons.

Additional examples of transgenes encoding therapeutic proteins includeclotting factors, to treat hemophilia and other coagulation/clottingdisorders (e.g., Factor VIII, IX or X); growth factors (e.g., growthhormone, insulin-like growth factor-1, platelet-derived growth factor,epidermal growth factor, acidic and basic fibroblast growth factors,transforming growth factor-β, etc.), to treat growth disorders orwasting syndromes; and antibodies (e.g., human or humanized), to providepassive immunization or protection of a subject against foreign antigensor pathogens (e.g., H. Pylori), or to provide treatment of cancer,arthritis or cardiovascular disease.

Additional transgenes encoding a therapeutic protein include cytokines,interferons (e.g., interferon (INF), INF-α 2b and 2a, INF-α N1, INF-β1b, INF-gamma), interleukins (e.g., IL-1 to IL-10), tumor necrosisfactor (TNF-α TNF-β), chemokines, granulocyte macrophage colonystimulating factor (GM-CSF), polypeptide hormones, antimicrobialpolypeptides (e.g., antibacterial, antifungal, antiviral, and/orantiparasitic polypeptides), enzymes (e.g., adenosine deaminase),gonadotrophins, chemotactins, lipid-binding proteins, filgastim(Neupogen), hemoglobin, erythropoietin, insulinotropin, imiglucerase,sarbramostim, tissue plasminogen activator (tPA), urokinase,streptokinase, neurite growth factor (NGF) phenylalanine ammonia lyase,brain-derived neurite factor (BDNF), neurite growth factor (NGF),phenylalanine ammonia lyase, thrombopoietin (TPO), superoxide dismutase(SOD), adenosine deamidase, catalase calcitonin, endothelian,L-asparaginase pepsin, uricase trypsin, chymotrypsin elastase,carboxypeptidase lactase, sucrase intrinsic factor, calcitoninparathyroid hormone (PTH)-like, hormone, soluble CD4, and antibodiesand/or antigen-binding fragments (e.g, FAbs) thereof (e.g., orthocloneOKT-e (anti-CD3), GPIIb/IIa monoclonal antibody).

The transgenes described herein are particular applications of theinvention but are not intended to limit it. In this regard, the skilledartisan could readily envision additional transgenes transcribed intotherapeutic antisense or encode therapeutic polypeptides.

Target cells include mucosal cells or cells not normally present in themucosum that can or have been adapted for growth in mucosum. As usedherein, the terms “mucosa” or “mucosal,” when used in reference to acell, means a cell that can grow in mucosa. Mucosal cells include, forexample, those cells which are normally found in animal mucosa, such asa cell of the gut (e.g., mouth (tongue and buccal tissue), esophagus,and stomach, small and large intestine, rectum, anus), the respiratorytract, the lungs and nasopharynx and other oral cavities (e.g., vagina).Thus, a mucosal cell refers to the various cell types that normallyreside in the aforementioned regions including stem cells or othermultipotent or pluripotent cells that differentiate into the variousmucosal cell types. Particular examples of mucosal cells includeendocrine cells, such as K cells, L-cells, S-cells, G-cells, D-cells,I-cells, Mo-cells, Gr-cells and entero-endocrine cells. Endocrine cellsare generally characterized by their ability to secrete a synthesizedprotein into the blood in response to a signal or stimuli (a“secretagogue”). Non-endocrine mucosal cells include epithelial cellswhich line the outer surface of most mucosal tissue, mucous cells,villus cells, columnar cells, stromal cells and Paneth cells.Non-endocrine cells are generally not known to secrete a synthesizedprotein into the blood in response to a signal or stimuli.

The finding that gut K cells can function as surrogate cells forproducing appropriately regulated physiologic levels of insulin inanimals indicates a mode of therapy for diabetes, freeing subjects frominsulin injections and reducing or even eliminating the associateddebilitating complications. As there are possibly billions of K cellsare present in the human gut (Sandström O., El-Salhy M., Mech. AgeingDev. 108:39 (1999)), regulated insulin secretion from a fraction ofthese cells may be sufficient to achieve therapeutic benefit, includingameliorating symptoms and complications associated with diabetes.

The gut is the largest endocrine organ in the body capable of producingvast quantities of proteins and contains rapidly renewing tissue inwhich the dividing cells are accessible. Target cells, such as K cellsand stem cells, are predominantly located in the upper gut which isreadily accessible to non-invasive gene therapy techniques. Thus,non-invasive techniques like oral formulations, endoscopic procedures,or a modified feeding tube allow the deployment of vectors thatfacilitate integration of the transgene into the host genome. Vectorshave already been developed that deliver genes to cells of theintestinal tract, including the stem cells (Croyle et al., Gene Ther.5:645 (1998); S. J. Henning, Adv. Drug Deliv. Rev. 17:341 (1997), U.S.Pat. Nos. 5,821,235 and 6,110,456). Many of these vectors have beenapproved for human studies. Therefore, gut cells, such as K cells, thatsecrete a protein, such as insulin, leptin, glucagon antagonist, GLP-1,GLP-2, Ghrelin, cholecystokinin, growth hormone, clotting factors,antibody, among others, in a regulatable fashion is a means with whichto treat diabetes, obesity, growth deficiency and other disorderstreatable by producing a protein in mucosal tissue.

A partial list of several types of gut endocrine cells, proteinssecreted by the cells in response to particular nutrients(“secretagogues”) and exemplary functions are shown in Table 2. Theproteins, endocrine cells and nutrients are all applicable in theinvention.

TABLE 2 CELL CELL PEPTIDE TYPE LOCATION FUNCTION SECRETAGOGUES GastrinG-cells Gastric Antrum increase acid Amino acids (stomach) secretionSomatostatin D-cells GI Tract reduce gut Intra-luminal acid, freepeptide release fatty acids, hormones (paracrine inhibitor)Glucose-dependent K cells Upper small Pancreatic Acid, bile salts, fattyInsulinotropic intestine bicarbonate acids Polypeptide secretion, reducegastric acid release Glucagon-like L-cells Lower small Increase Glucose,fat peptide-1 intestine insulin secretion, decrease gastric acid releaseGlucagon-like L-cells Lower small increase Glucose, fat peptide-2intestine mucosal proliferation Cholecystokinin I-cells Upper smallincrease gall Amino acids, fatty bladder acids contraction & pancreaticenzyme secretion Motilin Mo-cells Upper small Increase Cyclic release,meals intestine Gastric motility Ghrelin Gr-cells Stomach and orexigenicyet to be elucidated intestine

As used herein, the term “cultured,” when used in reference to a cell,means that the cell is grown in vitro. A particular example of such acell is a cell isolated from a subject, and grown or adapted for growthin tissue culture. Another example is a cell genetically manipulated invitro, and transplanted back into the same or a different subject. Theterm “isolated,” when used in reference to a cell, means a cell that isseparated from its naturally occurring in vivo environment. An exampleof an isolated cell would be a mucosal cell obtained from a subject suchas a human. “Cultured” and “isolated” cells may be manipulated by thehand of man, such as genetically transformed. These terms include anyprogeny of the cells, including progeny cells that may not be identicalto the parental cell due to mutations that occur during cell division.The terms do not include an entire human being.

The target mucosal cell may be present in a mucosal tissue or organ of asubject, such as that of the gut (e.g., intestine). Thus, one way inwhich to introduce the protein in the subjects' mucosum to achievetherapy is to intracellularly deliver a polynucleotide, including anexpression control element, in operable linkage with a nucleic acidencoding the protein into cells present in the mucosum of the subject.Alternatively, the mucosal cell can be isolated from an appropriatetissue of a subject, transfected with the transgene and introduced(transplanted) into a tissue (mucosal or other) of a subject. Thus,another way in which to introduce the protein into the subject toachieve therapy is to transfect a polynucleotide, including anexpression control element, in operable linkage with a nucleic acidencoding the protein into cultured mucosal cells, followed by implantingthe transformed cells or progeny into the subject.

Mucosal cells transfected with a transgene include endocrine andnon-endocrine cell lines that grow in-culture. For example, atransformed cell of gut origin or lineage, such as an STC-1 or GTC-1cell, can be implanted into a tissue of a subject. Mucosal cellstransfected with a transgene, in vitro, ex vivo or in vivo includeendocrine cells (e.g., K-cell, L-cell, G-cell, D-cell, S-cell, I-cell orMo-cell, Gr-cell) and non-endocrine epithelial, columnar, stromal,villus, Panth, stem cells or other cell types typically present inmucosal tissue of an animal.

The target cell may also be a non-mucosal cell (endocrine ornon-endocrine) which can grow or adapted for growth in mucosum or othertissue (even for a limited time, e.g. days or months). For example, acell may be obtained from a non-mucosal tissue of a subject, transformedwith a transgene or polynucleotide, and then transplanted into a tissueof subject (the same or different subject) in order to effect treatmentwhen the transcribed antisense or encoded protein is produced.Alternatively, a primary cell isolate or an established non-mucosal cellline can be transformed with a transgene or polynucleotide, and thentransplanted into a mucosal tissue of a subject.

Thus, to produce an isolated or cultured mucosal cell of the invention,the mucosal cell may be obtained from a tissue or organ of thegastrointestinal tract of a subject, for example. The mucosal cell canthen be transfected with the transgene by conventional nucleic acidtechniques and propagated. For example, intestinal stem cells can beisolated and then cultured and transfected in vitro (Booth, C. et al.,Exp. Cell Res. 241:359 (1999); Kawaguchi, A. L. et al., J. Pediatr.Surg. 33:559 (1998)). Cells that contain or express the transgene can beidentified using conventional methods, such as Southern, Northern orWestern blots, alone or in combination with selection using a selectablemarker. Transformed cells can then be re-introduced(transplanted/implanted) into the same or a different tissue of the sameor a different subject from which they were originally obtained.

If desired, target mucosal endocrine cells may contain multipletransgenes (i.e., two or more). In this way, expression of differentproteins encoded by the transgenes can provide an additive orsynergistic effect and, in turn, a therapeutic benefit greater thanexpression of either protein alone. In addition, if the two transgenesare linked to different expression control elements, or secretion of thetwo encoded polypeptides are regulated by different signals or stimuli(e.g., two different nutrients), the proteins can be produced eitherindependently of each other or in combination (when both of thedifferent nutrients are provided). For example, two transgenes, oneencoding GLP-1 and the other encoding insulin, can be constructed inwhich production is controlled by two different signals, such as glucoseand a drug, respectively. Glucose stimulates production of GLP-1 (eitherby stimulating transcription or secretion, as discussed herein) whereasthe drug stimulates production of insulin (either by stimulatingtranscription or secretion, as discussed herein). Addition of the drugto stimulate production of insulin (again, either by stimulatingtranscription or secretion) and addition of glucose can stimulateproduction of GLP-1; increased amounts of drug or glucose could induceeven greater amounts of insulin or GLP-1 production. Production of bothinsulin and GLP-1 by addition of the drug with a meal (containingglucose) may provide an even greater therapeutic benefit, especially forsubjects suffering from severe diabetes, for example. Accordingly, theinvention further includes mucosal cells containing multiple transgenesand methods of producing and using them.

Thus, in accordance with the invention, there are provided mucosalcell(s) that produces a protein regulatable by a nutrient, whereexpression of the protein is conferred by a transgene comprising anexpression control element in operable linkage with a nucleic acidencoding the protein. In one embodiment, the mucosal cell is anendocrine cell (e.g., a K-cell). In another embodiment, the mucosal cellis a non-endocrine cell. In yet another embodiment, the mucosal cell isa stem cell, or a multipotent or pluripotent progenitor cell. In anadditional embodiment, the expression control element confersnutrient-regulatable expression. In one aspect, the nutrient-regulatableelement comprises a gut endocrine promoter (e.g., a GIP promoter). Instill another embodiment, the nutrient increases secretion of a proteinencoded by the nucleic acid. In yet another embodiment, the nucleic acidencodes a therapeutic polypeptide (e.g., insulin, leptin, glucagon-likepeptide-1, glucagon-like peptide-2, a glucagon antagonist,cholecystokinin, a growth hormone, a clotting factor, an antibody, amongothers). In yet another embodiment, the mucosal cell includes two ormore transgenes.

The polynucleotides, including an expression control element, inoperable linkage with a nucleic acid, can be introduced for stableexpression into cells of a whole organism. Such organisms includingtransgenic animals, are useful for studying the effect of mucosalprotein production in a whole animal and therapeutic benefit. Forexample, as described herein, production of insulin in the gut of atransgenic mouse protects the animal from developing diabetes and fromglucose intolerance after destruction of pancreatic β-cells. Micestrains that develop or are susceptible to developing a particulardisease (e.g., diabetes, degenerative disorders, cancer, etc.) are alsouseful for introducing therapeutic proteins as described herein in orderto study the effect of therapeutic protein expression in the diseasesusceptible mouse. Transgenic and genetic animal models that aresusceptible to particular disease or physiological conditions are knownin the art and are appropriate targets for expressing therapeuticproteins in gut.

Thus, in accordance with the invention, there are provided non-humantransgenic animals that produce a protein in mucosal tissue, productionnot naturally occurring in mucosal tissue of the animal, productionconferred by a transgene present in somatic or germ cells of the animal.In one embodiment, the transgene comprises a polynucleotide, includingan expression control element in operable linkage with a nucleic acidencoding a therapeutic polypeptide (e.g., insulin, leptin, GLP-1, GLP-2,Ghrelin, CCK, glucagon antagonist, growth hormone, clotting factor,antibody, among others.) In another embodiment, the transgenic animal isa mouse. In yet another embodiment, expression of the therapeuticpolypeptide in the mucosal tissue of the animal is responsive to anutrient. In still another embodiment, secretion of therapeuticpolypeptide in mucosal tissue is increased by a nutrient. In a furtherembodiment, expression of the therapeutic polypeptide in mucosal tissueis increased by a nutrient (i.e., the expression control elementcontrolling expression of insulin comprises a nutrient-inducibleelement). In an additional embodiment, the nutrient-regulatable elementcomprises a glucose-inducible promoter (e.g., a glucose-dependentinsulinotropic polypeptide promoter). In additional embodiments, themucosal tissue is a tissue or organ of the gastrointestinal tract (e.g.,intestine) or gut, and includes endocrine cells. In a furtherembodiment, isolated cells of the invention transgenic animals thatexpress the therapeutic polypeptide are provided.

The term “transgenic animal” refers to an animal whose somatic or germline cells bear genetic information received, directly or indirectly, bydeliberate genetic manipulation at the subcellular level, such as bymicroinjection or infection with recombinant virus. The term“transgenic” further includes cells or tissues (i.e., “transgenic cell,”“transgenic tissue”) obtained from a transgenic animal geneticallymanipulated as described herein. In the present context, a “transgenicanimal” does not encompass animals produced by classical crossbreedingor in vitro fertilization, but rather denotes animals in which one ormore cells receive a nucleic acid molecule. Invention transgenic animalscan be either heterozygous or homozygous with respect to the transgene.Methods for producing transgenic animals, including mice, sheep, pigsand frogs, are well known in the art (see, e.g., U.S. Pat. Nos.5,721,367, 5,695,977, 5,650,298, and 5,614,396) and, as such, areadditionally included.

In accordance with the invention, there are provided methods of treatinga subject having, or at risk of having, a disorder treatable byproducing a therapeutic protein in a mucosal tissue. In one embodiment,a method of the invention includes contacting mucosal tissue cells inthe subject transformed with a polynucleotide (in vitro, ex vivo or invivo) comprising an expression control element in operable linkage witha nucleic acid encoding the therapeutic protein with a nutrient thatinduces production of the protein in an amount effective to treat thedisorder. In another embodiment, a method of the invention includesproducing a therapeutic protein in a mucosal tissue of the subject byimplanting one or more transformed mucosal cells (in vitro or ex vivo)that produce the protein into the subject's tissue in an amounteffective for treating the disorder.

Disorders treatable by a method of the invention include a hyperglycemiccondition, such as insulin-dependent (type 1) or -independent (type 2)diabetes, as well as physiological conditions or disorders associatedwith or that result from the hyperglycemic condition. Thus,hyperglycemic conditions treatable by a method of the invention alsoinclude a histopathological change associated with chronic or acutehyperglycemia (e.g., diabetes). Particular examples include degenerationof pancreas (β-cell destruction), kidney tubule calcification,degeneration of liver, eye damage (diabetic retinopathy), diabetic foot,ulcerations in mucosa such as mouth and gums, excess bleeding, delayedblood coagulation or wound healing and increased risk of coronary heartdisease, stroke, peripheral vascular disease, dyslipidemia, hypertensionand obesity.

Thus, in various methods of the invention, a mucosal cell that producesinsulin or a functional subsequence of insulin in response to glucose,is useful for increasing insulin, decreasing glucose, improving glucosetolerance, treating a hyperglycemic condition (e.g., diabetes) or fortreating a physiological disorders associated with or resulting from ahyperglycemic condition. Such disorders include, for example, diabeticneuropathy (autonomic), nephropathy (kidney damage), skin infections andother cutaneous disorders, slow or delayed healing of injuries or wounds(e.g., that lead to diabetic carbuncles), eye damage (retinopathy,cataracts) which can lead to blindness, diabetic foot and acceleratedperiodontitis. Such disorders also include increased risk of developingcoronary heart disease, stroke, peripheral vascular disease,dyslipidemia, hypertension and obesity.

As used herein, the term “hyperglycemic” or “hyperglycemia,” when usedin reference to a condition of a subject, means a transient or chronicabnormally high level of glucose present in the blood of a subject. Thecondition can be caused by a delay in glucose metabolization orabsorption such that the subject exhibits glucose intolerance or a stateof elevated glucose not typically found in normal subjects (e.g., inglucose-intolerant subdiabetic subjects at risk of developing diabetes,or in diabetic subjects). Fasting plasma glucose (FPG) levels fornormoglycemia are less than about 110 mg/dl, for impaired glucosemetabolism, between about 110 and 126 mg/dl, and for diabetics greaterthan about 126 mg/dl.

Disorders treatable by producing a protein in a mucosal tissue alsoinclude obesity or an undesirable body mass. Leptin, cholecystokinin andGLP-1 decrease hunger, increase energy expenditure, induce weight lossor provide normal glucose homeostasis. Thus, in various embodiments, amethod of the invention for treating obesity or an undesirable bodymass, or hyperglycemia, includes contacting mucosal tissue cells havinga transgene encoding leptin, cholecystokinin or GLP-1 with a nutrient soas to produce the protein in an amount effective to treat obesity or anundesirable body mass. Disorders treatable also include those typicallyassociated with obesity, for example, abnormally elevated serum/plasmaLDL, VLDL, triglycerides, cholesterol, plaque formation leading tonarrowing or blockage of blood vessels, increased risk ofhypertension/stroke, coronary heart disease, etc.

As used herein, the term “obese” or “obesity” refers to a subject havingat least a 30% increase in body mass in comparison to an age and gendermatched normal subject. “Undesirable body mass” refers to subjectshaving 1%-29% greater body mass than a matched normal subject as well assubjects that are normal with respect to body mass but who wish todecrease or prevent an increase in their body mass.

The term “subject” refers to an animal. Typically, the animal is amammal, however, any animal having mucosal tissue, such as gut, isencompassed by the term. Particular examples of mammals are primates(humans), dogs, cats, horses, cows, pigs, and sheep. Subjects includethose having a disorder, e.g., a hyperglycemic disorder, such asdiabetes, or subjects that do not have a disorder but may be at risk ofdeveloping the disorder, e.g., subdiabetic subjects having FPG levelsbetween about 110 and 126 mg/dl. Subjects at risk of developing adisorder include, for example, those whose diet may contribute to or beassociated with development of diabetes or obesity, as well as thosewhich may have a family history or genetic predisposition towardsdevelopment of diabetes or obesity. Subjects also include apparentlynormal subjects, for example, those who wish to lose weight but are notconsidered to be obese or have greater than normal body mass.

A partial list of therapeutic proteins and target diseases is shown inTable 3.

TABLE 3 LEAD COMPOUNDS TARGET DISEASE FUNCTION THERAPEUTIC EFFECTInsulin Diabetes Insulin replacement Improve glucose toleranceDelay/prevent diabetes Glucagon Diabetes Reduce endogenous Improveglucose tolerance antagonists glucose production GLP-1 Diabetes ObesityStimulate growth of β- Improve glucose tolerance cells, improve insulinInduce weight loss sensitivity, suppress appetite Leptin ObesityDiabetes Appetite suppression Induce weight loss and improvement ofImprove glucose tolerance insulin sensitivity CCK Obesity Appetitesuppression Induce weight loss Growth hormone GH deficiencies, GHreplacement Improve growth (GH) wasting and anti- aging Clotting factorsHemophilia Clotting factors Improve clotting time replacementTherapeutic Infections Pathogen Prevent infections or human Cancersneutralization or transplant rejections monoclonal immune modulationsantibodies

Treatment generally results in reducing or preventing the severity orsymptoms of the condition in the subject, i.e., an improvement in thesubject's condition or a “therapeutic effect.” Therefore, treatment canreduce the severity or prevent one or more symptoms of the condition oran associated disorder, inhibit progression or worsening of thecondition or an associated disorder, and in some instances, reverse thecondition or an associated disorder. Thus, in the case of ahyperglycemic condition, for example, treatment can reduce bloodglucose, improve glucose tolerance, provide normal glucose homeostasis,or prevent, improve, or reverse a histopathological change associatedwith or that results from the hyperglycemic condition.

Improvement of a histopathological change associated with ahyperglycemic condition includes, for example, preventing further orreducing kidney tubule calcification, decreasing or arrestingretinopathy or cataracts, decreasing wound or injury healing time,reducing diabetic foot, preventing or reducing acceleratedperiodontitis, or decreasing the risk of developing coronary heartdisease, stroke, peripheral vascular disease, dyslipidemia, hypertensionand obesity. Improvement in obesity can include, for example, areduction of body mass or an improvement in an associated disorder, suchas a decrease in cholesterol, LDL or VLDL levels, a decrease in bloodpressure, a decrease in intimal thickening of the blood vesselassociated with high fat diet, a decrease in resting heart rate, anincrease in lung capacity, etc. Improvement in a bleeding disorder, suchas hemophilia can induce, for example, decreased clotting time orfrequency/duration of bleeding episodes.

As used herein, the term “ameliorate” means an improvement in thesubject's condition, a reduction in the severity of the condition, or aninhibition of progression or worsening of the condition. In the case ofa hyperglycemic condition (e.g., diabetes), for example, an improvementcan be a decrease in blood glucose, an increase in insulin, animprovement in glucose tolerance, or glucose homeostasis. An improvementin a hyperglycemic condition also can include improved pancreaticfunction (e.g., inhibit or prevent β-islet cell destruction), a decreasein a pathology associated with or resulting from the condition, such asan improvement in histopathology of an affected tissue or organ, as setforth herein. In the case of obesity, for example, an improvement can bea decrease in weight gain, a reduction of body mass or an improvement ina conditions associated with obesity, as set forth herein (e.g.,reduction of blood glucose, cholesterol, LDL or VLDL levels, a decreasein blood pressure, a decrease in intimal thickening of the blood vessel,etc.). In the case of hemophilia or other bloodcoagulation/clotting/bleeding disorders, an improvement can reduce thefrequency or duration of bleeding episodes or hemorrhage. Improvementslikewise include chronic disorders associated with bloodcoagulation/clotting/bleeding associated disorders such as a reductionin neurological problems, crippling tissue and joint damage, forexample.

The doses or “effective amount” for treating a subject are preferablysufficient to ameliorate one, several or all of the symptoms of thecondition, to a measurable or detectable extent, although preventing orinhibiting a progression or worsening of the disorder or condition, or asymptom, is a satisfactory outcome. Thus, in the case of a condition ordisorder treatable by producing a protein in a mucosal tissue, theamount of protein produced, or transplanted cell(s) sufficient toameliorate a condition treatable by a method of the invention willdepend on the condition and the desired outcome and can be readilyascertained by the skilled artisan. Appropriate amounts will depend uponthe condition treated, the therapeutic effect desired, as well as theindividual subject (e.g., the bioavailability within the subject,gender, age, etc.). For example, a partial restoration of normal glucosehomeostatsis in a subject can reduce the frequency for insulininjection, even though complete freedom from insulin injection has notresulted.

The effective amount can be ascertained by measuring relevantphysiological effects. For example, in the case of diabetes or otherhyperglycemic condition, a decrease in blood glucose or an improvementin glucose tolerance test can be used to determine whether the amount ofinsulin, or cell(s) expressing insulin transplanted into the animalmucosa, is effective to treat the hyperglycemic condition. For example,an amount reducing FPG from 126 mg/dl to 120, 115, 110, or less is aneffective amount. In the case of obesity or an undesirable body mass, adecrease in the subjects' mass, a decrease in meal size or caloriccontent of a meal, increased satiety for a given meal size, anddecreases in serum/plasma levels of lipid, cholesterol, fatty acids, LDLor VLDL all can be effective amounts for ameliorating obesity or anundesirable body mass of a subject. In the case of hemophilia, aneffective amount is an amount which reduces clotting time or frequencyor duration of bleeding episodes in a subject.

The methods of the invention for treating a subject are applicable forprophylaxis to prevent a condition in a subject, such as a hyperglycemiccondition or an associated disorder, or development of obesity or anincreased body mass. Alternatively, the methods can be practicedfollowing treatment of a subject as described herein. For example,following treatment and a reduction of body mass to the desired weight,leptin, GLP-1 or CCK can be periodically produced by mucosal cells, asdescribed herein, in order to suppress appetite, decrease mealconsumption, etc. thereby maintaining desired body weight.

The methods of the invention for treating a subject also can besupplemented with other forms of therapy. Supplementary therapiesinclude drug treatment, a change in diet (low sugar, fats, etc.)surgical resection, transplantation, radiotherapy, etc. For example, amethod of the invention for treating a hyperglycemic condition can beused in combination with drugs or other pharmaceutical formulations thatincrease insulin or lower glucose in a subject. Drugs for treatingdiabetes include, for example, biguanides and sulphonylureas (e.g.,tolbutamide, chlorpropamide, acetohexamide, tolazamide, glibenclamideand glipizide). Appetite suppression drugs are also well known and canbe used in combination with the methods of the invention. Supplementarytherapies can be administered prior to, contemporaneously with orfollowing the invention methods of treatment. The skilled artisan canreadily ascertain therapies that may be used in a regimen in combinationwith the treatment methods of the invention.

As a method of the invention can include in vivo delivery, such as apolynucleotide comprising an expression control element in operablelinkage with a nucleic acid into mucosal cells of a subject, in order toproduce an encoded protein in the subject, for example, expressionsystems further include vectors specifically designed for in vivodelivery. Vectors that efficiently deliver genes to cells of theintestinal tract (e.g., stem cells) have been developed and arecontemplated for use in delivering the polynucleotides into mucosalcells (see, e.g., U.S. Pat. Nos. 5,821,235, 5,786,340 and 6,110,456;Croyle, M. A. et al., Gene Ther. 5:645 (1998); Croyle, M. A. et al.,Pharm. Res. 15:1348 (1998); Croyle, M. A. et al., Hum. Gene Ther. 9:561(1998); Foreman, P. K. et al., Hum. Gene Ther. 9:1313 (1998); Wirtz, S.et al., Gut 44:800 (1999)). Adenoviral and adeno-associated viralvectors suitable for gene therapy are described in U.S. Pat. Nos.5,700,470, 5,731,172 and 5,604,090. Additional vectors suitable for genetherapy include herpes simplex virus vectors (see, e.g., U.S. Pat. No.5,501,979), retroviral vectors (see, e.g., U.S. Pat. Nos. 5,624,820,5,693,508 and 5,674,703; and WO92/05266 and WO92/14829), bovinepapilloma virus (BPV) vectors (see, e.g., U.S. Pat. No. 5,719,054),CMV-based vectors (see, e.g., U.S. Pat. No. 5,561,063) and parvovirus,rotavirus and Norwalk virus vectors. Lentiviral vectors are useful forinfecting dividing as well as non-dividing cells (see, e.g., U.S. Pat.No. 6,013,516).

Introduction of nucleic acid and polypeptide in vitro, ex vivo and invivo can also be accomplished using other techniques. For example, apolynucleotide comprising an expression control element in operablelinkage with a nucleic acid encoding a protein can be incorporated intoparticles or a polymeric substance, such as polyesters, polyamine acids,hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers. A polynucleotide can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, forexample, by the use of hydroxymethylcellulose or gelatin-microcapsules,or poly (methylmethacrolate) microcapsules, respectively, or in acolloid drug delivery system. Colloidal dispersion systems includemacromolecule complexes, nano-capsules, microspheres, beads, andlipid-based systems, including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The use of liposomes for introducing variouscompositions, including polynucleotides, is known to those skilled inthe art (see, e.g., U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and4,975,282). A carrier comprising a natural polymer, or a derivative or ahydrolysate of a natural polymer, described in WO 94/20078 and U.S. Pat.No. 6,096,291, is suitable for mucosal delivery of molecules, such aspolypeptides and polynucleotides. Piperazine based amphilic cationiclipids useful for gene therapy also are known (see, e.g., U.S. Pat. No.5,861,397). Cationic lipid systems also are known (see, e.g., U.S. Pat.No. 5,459,127). Accordingly, vector (viral and non-viral, e.g., nakedDNA) and non-vector means of delivery into mucosal cells or tissue, invitro, in vivo and ex vivo can be achieved and are contemplated.

As the methods of the invention can include contacting a mucosal cell(s)present in a subject with a polynucleotide, the present invention alsoprovides “pharmaceutically acceptable” or “physiologically acceptable”formulations in which a transgene or therapeutic polypeptide areincluded. Such formulations can be administered ex vivo or in vivo to asubject in order to practice the treatment methods of the invention, forexample.

As used herein, the terms “pharmaceutically acceptable” and“physiologically acceptable” refer to carriers, diluents, excipients andthe like that can be administered to a subject, preferably withoutproducing excessive adverse side-effects (e.g., nausea, abdominal pain,headaches, etc.). Such preparations for administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions.

Pharmaceutical formulations can be made from carriers, diluents,excipients, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with administration to a subject. Such formulations canbe contained in a tablet (coated or uncoated), capsule (hard or soft),microbead, emulsion, powder, granule, crystal, suspension, syrup orelixir. Supplementary active compounds and preservatives, among otheradditives, may also be present, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.

A pharmaceutical formulation can be formulated to be compatible with itsintended route of administration. Thus, pharmaceutical formulationsinclude carriers, diluents, or excipients suitable for administration byroutes including intraperitoneal, intradermal, subcutaneous, oral (e.g.,ingestion or inhalation), intravenous, intracavity, intracranial,transdermal (topical), parenteral, e.g. transmucosal and rectal.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following: a sterile diluentsuch as water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical formulations suitable for injection include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. Fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride can beincluded in the composition. Prolonged absorption of injectableformulations can be achieved by including an agent that delaysabsorption, for example, aluminum monostearate or gelatin.

For oral administration, a composition can be incorporated withexcipients and used in the form of tablets, troches, or capsules, e.g.,gelatin capsules. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included in oral formulations. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or flavoring.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. For example, a time delay material such as glycerylmonostearate or glyceryl stearate alone, or in combination with a wax,may be employed.

Additional formulations include biodegradable or biocompatible particlesor a polymeric substance such as polyesters, polyamine acids, hydrogel,polyvinyl pyrrolidone, polyanhydrides, polyglycolic acid,ethylene-vinylacetate, methylcellulose, carboxymethylcellulose,protamine sulfate, or lactide/glycolide copolymers,polylactide/glycolide copolymers, or ethylenevinylacetate copolymers inorder to control delivery of an administered composition. Methods forpreparation of such formulations will be apparent to those skilled inthe art. The materials can also be obtained commercially from AlzaCorporation and Nova Pharmaceuticals, Inc., for example.

The rate of release of a composition can be controlled by altering theconcentration or composition of such macromolecules. For example, thecomposition can be entrapped in microcapsules prepared by coacervationtechniques or by interfacial polymerization, for example, by the use ofhydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Additional pharmaceutical formulations appropriate for administrationare known in the art and are applicable in the methods and compositionsof the invention (see, e.g., Remington's Pharmaceutical Sciences (1990)18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12thed., Merck Publishing Group, Whitehouse, N.J.; and PharmaceuticalPrinciples of Solid Dosage Forms, Technonic Publishing Co., Inc.,Lancaster, Pa., (1993)).

The mucous or endothelial lining of the mucosal tissue may be removed orotherwise prepared prior to administration, for example, usingpenetrants or other barrier penetration enhancers. Such penetrantsappropriate to the barrier to be permeated are generally known in theart, and include, for example, for transmucosal administration,incubation with N-acetyl-cysteine (Nakanishi et al. Chem Pharm Bull(Tokyo) 40:1252 (1992), Meaney and O'Driscoll Eur J Pharm Sci. 8:167(1999); hydrolysis of intestinal mucins by purified Sigma 1 protein andinfectious subviral particles (Bisaillon et al. J Mol. Biol. 286:759(1999); desialation (Slomiany et al. Gen Pharmacol. 27:761 (1996);(Hirmo et al. FEMS Immunol Med. Microbiol. 20:275 (1998); desulphationby H. pylori glycosulfatase (Slomiany et al. Am J. Gastroenterol.87:1132 (1992); desialation by neuraminidase (Hanski et al. Cancer Res.51:5342 (1991)); disulphide bond breakage by β-mercaptoethanol(Gwozdzinski et al. Biochem Int. 17:907 (1988); deglycosylation withspecific exoglycosidases such as fucosidase, β-galactosidase,N-acetyl-galactosaminidase, β-N-acetyl hexososaminidase, andneuraminidase (Slomiany et al. Biochem Biophys Res Commun. 142:783(1987); acid removal of by 0.4 N HCl (Ruggieri et al. Urol Res. 12:199(1984), Davis C. P. and Avots-Avotins A. E. Scan Electron Microsc. (Pt2):825-30 (1982), Parsons et. al. Am J Pathol. 93:423 (1978)), amongothers. Mucosal administration can also be accomplished through the useof nasal sprays or suppositories. For administration by inhalation, theformulation can be delivered via a pump or an aerosol spray from adispenser or pressured container that contains a suitable propellant,e.g., a gas such as carbon dioxide, or a nebulizer.

The number of stem cells can be increased by exposure to cytotoxicagents and growth factors. For example, irradiation of the small gutincreases the number clonogenic/stem cells (Roberts S. A. Radiat. Res.141:303 (1995); Cai W. B. et. al. Intl. J. Radiat. Biol. 71:145 (1997)).In addition, treatment with GLP-2, epidermal growth factor, TGF-α,insulin-like growth factors, interleukins, among others, have been shownto promote the growth of mucosal cells (Potten C. S. Int. J. Exp. Path78:219 (1997)). In this way, additional target cells can be producedthereby increasing transformation efficiency and subsequent regulatedprotein production by transformed cells.

Endoscopes, cannulas, intubation tubes, catheters and the like can beused to deliver the formulation to various parts of the gut of asubject. This allows effective delivery and targeting of vectors toparticular areas of the gut.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All publications, patents and other references cited herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a mucosal cell” includes a plurality of suchcells and reference to “a polynucleotide comprising an expressioncontrol element in operable linkage with a nucleic acid” includesreference to one or more such constructs, and so forth.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

Example I

This example describes the establishment of a gut endocrine cell lineuseful for studying regulated insulin production and for targetinginsulin expression in vivo. This example also describes construction ofa human insulin gene expression vector.

A GIP-expressing cell line was established to investigate whether theGIP promoter is effective in targeting insulin gene expression to Kcells. This cell line was cloned from the murine intestinal cell lineSTC-1, a mixed population of gut endocrine cells (Rindi et. al., Am. J.Pathol. 136:1349 (1990)). K cells in the mixed population were visuallyidentified by transfection of a green fluorescent protein expressionplasmid driven by ˜2.5 Kb of the rat GIP promoter. The rat GIP promoterwas obtained from a rat genomic λDASH library (Stratagene) by plaquehybridization with the rat GIP cDNA clone as described previously(Boylan et. al., J. Biol. Chem. 273:17438 (1997)) and subcloned into thepromoterless pEGFP-1 plasmid (Clontech). The resulting reporter vectorwas transfected into STC-1 cells (D. Drucker, University of Toronto)using Lipofectamine (GIBCO). Cells were dispersed with Trypsin/EDTA andfluorescent cells expressing EGFP were double hand-picked and placedinto individual dishes for clonal expansion (FIG. 1).

Following clonal expansion of the transiently fluorescent cells, cloneswere analyzed for the expression of GIP mRNA by northern blotting. Inbrief, total RNA from GTC-1 and STC-1 cells was isolated with Trizol(Gibco) according to manufacturer's instructions. Total cell RNA (20 ug)from each sample was electrophoretically separated and transferred tonylon membrane. Hybridization was performed with radiolabeled 660 bpEcoRI fragment of the rat GIP cDNA that was random-primed with[α-³²P]dCTP. Following hybridization, membranes were washed and exposedto x-ray film. The level of GIP mRNA in one clone (GIP Tumor Cells;GTC-1) was ˜8-fold higher than in the parental heterogeneous STC-1 cells(FIG. 2).

In order to determine if GTC-1 cells correctly process human genomicpreproinsulin, an insulin expression construct in which the insulin genelinked to the 3′ end of the rat GIP promoter (FIG. 3, GIP/Ins) wastransfected into these cells.

To construct the human insulin/GIP expression plasmid, a ˜2.5 Kb portionof the rat GIP promoter was inserted into pGLBH as discussed above(Boylan et al., J. Biol. Chem. 273:17438 (1997)). Human insulin cDNA,which comprises ˜16 Kb of the genomic sequence extending fromnucleotides 2127 to 3732 including the native polyadenylation site, wasexcised from pBR322 (ATCC No. 57399) by digestion with BamHI and ligatedinto the BglII site of the GIP containing pGLBH construct. Theexpression construct is shown in FIG. 3.

Total RNA was isolated from GIP/Ins-transfected and non-transfectedcells and human islets (Trizol, GIBCO). Five μg of the RNA isolated wasreversed transcribed with oligo-dT primer using superscript II reversetranscriptase (GIBCO). Two μl of the cDNA product was amplified withhuman preproinsulin gene-specific primers (Primer 1 and 3, FIG. 3). Theresults indicate that human preproinsulin mRNA transcript was correctlyprocessed (FIG. 4, Top).

When the GIP/Ins construct was transfected into a β-cell (INS-1), liver(HepG2) and rat fibroblast (3T3-L1) cell line, little humanpreproinsulin mRNA was detectable. These observations indicate that theGIP promoter is cell specific and is likely to be effective in targetingtransgene expression to K cells in vivo.

Western blot analysis of GTC-1 cells to determine if processing enzymesfor converting proinsulin to mature insulin were present was thenperformed. In brief, GTC-1 cells were lysed in ice-cold RIPA buffer andsupernatants were assayed for total protein content using the Bradfordmethod. Cell lysate protein (50 μg) was fractionated on 10% SDS-PAGE andfractionated proteins were electroblotted onto nitrocellulose membranesand incubated with polyclonal antibodies recognizing PC1/3 and PC2 (Dr.Iris Lindberg, Louisiana State Medical Center). Membranes were washed,incubated with goat anti-rabbit antisera coupled to horseradishperoxidase (Amersham-Pharmacia) and developed with a chemiluminescencewestern blotting detection kit. The results indicate that the proproteinconvertases required for correct processing of proinsulin to matureinsulin (PC1/3 and PC2; Steiner, D. F., Curr. Opin. Chem. Biol. 2:31(1998)) were expressed in GTC-1 cells (FIG. 4, Bottom).

To confirm that proinsulin was appropriately processed, insulin andC-peptide levels in the cell culture media were measured (FIG. 5). BothC-peptide and insulin were detected in culture media collected fromGTC-1 cell transfected with the GIP/Ins plasmid. This result indicatesthat K cells are process proinsulin to mature insulin.

To confirm that production of human insulin from GTC-1 cells transfectedwith the GIP/Ins plasmid was glucose regulatable, insulin levels in thecell culture media under different concentrations of glucose wereassayed. In brief, 70-80% confluent GTC-1 cells in 12-well plates werefasted 2 hr in DMEM with 1.0 mM glucose and 1% Fetal calf serum (FCS).Cells were washed and then incubated in 0.5 mL of release media (DMEMplus 1% FCS with either 1.0 or 10.0 mM of glucose) for 2 hr. Medium wascollected after 2 hours for each condition and assayed using thehuman-specific insulin ELISA kit according to the supplier'sinstructions (ALPCO). Furthermore, release of insulin from these cellswas glucose-dependent (FIG. 6).

Example II

This example describes transgenic mice that produce insulin in responseto glucose.

Using the human insulin expression construct GIP/Ins described inExample I, the GIP/insulin fragment (˜4.1 Kb) was removed by digestionwith HindIII. Transgenic mice were generated by pronuclearmicroinjection of the ˜4.1 Kb transgene into fertilized embryos thatwere implanted into pseudopregnant females. Transgenic offspring wereidentified by Southern blot analysis. DNA from ear sections was digestedwith XhoI and Pvull (FIG. 3), electrophoretically separated, andtransferred to nylon membrane. For the detection of the transgene, a 416bp human insulin gene fragment encompassing intron 2 was amplified usingprimers 2 and 4 (FIG. 3). The PCR product was prepared as a probe byrandom labeling with [α-³²P] dCTP, and bands were detected byautoradiography. Southern analysis results were further confirmed by PCRamplification of the genomic DNA using primers 2 and 4. Positivefounders were outbred with wild-type FVB/N mice to establish transgeniclines (FIG. 8).

Transgenic mice tissues were examined for insulin expression. In brief,total RNA (50 μg) for each mouse stomach and duodenum, ileum, muscle,liver, spleen, kidney, fat, brain, lung, heart, bladder and testes werefractionated, transferred to a membrane and probed with a 333 base paircDNA fragment encompassing exons 1 and 2 and part of exon 3 of humanpreproinsulin gene. The analysis revealed that insulin was expressed inthe stomach and duodenum, but not in ileum, muscle, liver, spleen,kidney fat, brain, lung, heart, bladder or testes from the resultingtransgenic animals (FIG. 9).

To confirm insulin production in duodenum, RT-PCR analysis for insulinmRNA was performed. In brief, human proinsulin specific, forward 5%CCAGCCGCAGCCTTTGTGA-3′ and reverse 5′-GGTACAGCATTGTTCCACAATG-3′; mouseproinsulin specific, forward 5′-ACCACCAGCCCTAAGTGAT-3′ and reverse5′-CTAGTTGCAGTAGTTCTCCAGC-3′ primers used were. PCR conditions were asfollows: denaturation at 94° C. for 1 min, annealing at 50° C. for 1 minand extension at 72° C. for 1 min for 45 cycles. PCR products wereanalyzed on a 2% agarose gel and visualized by ethidium bromidestaining. The human- and mouse-specific primer sets yield 350 bp and 396bp products, respectively.

Insulin RNA was detected in the duodenum sample from the transgenic miceconfirming that insulin was not due to contamination from adjacent mousepancreas (FIG. 9). Cellular localization of insulin protein wasdetermined in tissue biopsies from transgenic mice utilizing antibody toinsulin. Insulin immunoreactivity was detected in distinct endocrinecells in sections from stomach of transgenic animals (FIG. 10).

The aforementioned results indicate that tissue distribution of insulinexpression in transgenic animals corresponds to the tissue expressionpattern of GIP (Tseng et al., Proc. Natl. Acad. Sci. USA 90:1992 (1993);Yeung et al., Mol. Cell. Endocrinol. 154:161 (1999)).

To determine whether the cells that expressed insulin were K cells,tissues were analyzed for immune-reactivity with GIP antisera. In brief,tissues were fixed in Bouin's solution overnight and embedded inparaffin. Tissue sections (5 μm thick) were mounted on glass slides. Forimmunohistochemistry, the avidin-biotin complex method was used withperoxidase and diaminobenzidine as the chromogen. Sections wereincubated with guinea pig anti-insulin (1:500; Linco Research, Inc.) ormouse anti-GIP (1:200; R. Pederson, University of British Columbia) for30 min and appropriate secondary antibodies for 20 min at roomtemperature. Biotinylated secondary antibodies were used forimmunohistochemistry, and fluorescein- or Cy3-conjugated secondaryantibodies were used for immunofluorescence. The results indicate thatinsulin-expressing cells were K cells due to co-expression ofimmunoreactive GIP (FIG. 10). These results confirm that human insulinproduction was effectively targeted to K cells in the gut of mice.

Example III

This example shows that insulin production in transgenic mice providednormal glucose homeostasis and protection from developing diabetes.Production of human insulin from gut K cells of transgenic mice is alsomeal regulated. This example also describes data showing that glucoseinducible insulin production by the transgenic mice provides glucosehomeostasis after destruction of pancreatic β cells.

Analysis of plasma human insulin levels in transgenic mice in responseto food intake was performed. In brief, plasma insulin levels weremeasured using the human-specific insulin ELISA kit (ALPCO) according tosupplier's instructions. This assay has <0.01% cross-reactivity withhuman proinsulin and C-peptide and does not detect mouse insulin. PlasmaC-peptide measurements were made with a rat/mouse C-peptide RIA kit(Linco). The assay displays no cross-reactivity with human C-peptide.

In pooled plasma samples collected after oral glucose challenge, insulinwas 39.0±9.8 pM (n=10, Mean±SEM) in transgenic and undetectable incontrols (n=5). To confirm that human insulin produced from K cells ismeal regulated, transgenic mice were fasted. Following a 40 hour fast,blood samples were collected via the tail vein. Animals were then refedwith a standard chow and blood samples were collected again 24 hr afterfood replacement.

As shown in FIG. 11A, fasting significantly reduced the circulatinghuman insulin in transgenic mice by more 40% (13.0±4.2 pM vs 7.6±2.3 pM,p<0.03). After food restriction, refeeding resulted in over 400%increase in circulating human insulin.

To evaluate the release kinetics of human insulin from gut K cells,fasted transgenic mice were fed either a mixed meal in the form of achow pellet (0.5 g) or an oral glucose challenge (3 mg/g body weight).As shown in FIG. 11B, both oral nutrient challenges promptly stimulatedthe release of human insulin from gut K cells by at least 20% within 30min. These results confirmed that insulin secretion from gut K cells isindeed meal-regulated.

Interestingly, levels of mouse C-peptide after an oral glucose load intransgenics were ˜30% lower than controls (227.1±31.5 pM vs 361.5±31.2pM, n=3 in each group, mean±SEM). This observation suggests that humaninsulin produced from the gut may have led to compensatorydown-regulation of endogenous insulin production.

The ability of human insulin production from gut K cells to protecttransgenic mice from diabetes was investigated. Streptozotocin (STZ), aβ-cell toxin, was administered to transgenic mice and age-matchedcontrols. In brief, Streptozotocin (200 mg/kg body weight) in citratebuffer was administered to 8 week old transgenic and age-matched controlmice via an intraperitoneal injection. At this dose of streptozotocin,mice typically display glucosuria within 3 days post injection.

In control animals, STZ treatment resulted in fasting hyperglycemia(26.2±1.52 mM, n=3, mean±SEM) and the presence of glucose in the urinewithin 3 to 4 days, indicating the development of diabetes. When leftuntreated these animals deteriorated rapidly and died within 7 to 10days. In contrast, neither glucosuria nor fasting hyperglycemia(9.52±0.67 mM, n=5, mean±SEM) was detected in transgenic mice for up tothree months after STZ treatment and they continued to gain weightnormally.

To determine if insulin production from K cells was able to maintainoral glucose tolerance in these mice despite the severe β-cell damage bySTZ, mice were challenged with an oral glucose load five days after STZtreatment. In brief, glucose was administered orally by feeding tube(1.5 g/kg body weight) as a 40% solution (wt/vol) to mice fasted for 14hr. Blood samples (40 μl) were collected from the tail vein of consciousmice at 0, 10, 20, 30, 60, 90, and 120 minutes following the glucoseload. Plasma glucose levels were determined by enzymatic, colorimetricassay (Sigma) and plasma insulin levels were measured usinghuman-specific insulin ELISA kit (ALPCO).

Control mice given STZ were severely hyperglycemic both before and afterthe glucose ingestion (FIG. 12). In contrast, STZ-treated transgenicmice had normal blood glucose levels and rapidly disposed of the oralglucose load as did normal age-matched control mice (FIG. 12).

To ensure that the STZ treatment effectively destroyed the β-cells inthese experimental animals, pancreatic sections from controls andSTZ-treated transgenic animals were immunostained for mouse insulin aspreviously described. The number of cell clusters positively stained formouse insulin was substantially lower in STZ-treated animals whencompared to sham-treated controls (FIG. 13). Total pancreatic content ofinsulin in STZ-treated transgenic mice was assessed by homogenizingpancreata and sonication at 4° C. in 2 mM acetic acid containing 0.25%BSA. After incubation for 2 hr on ice, tissue homogenates wereresonicated, centrifuged (8,000 g, 20 min) and supernatants were assayedfor insulin by radioimmunoassayonly. The results indicate that totalpancreatic content in STZ-treated transgenic mice was 0.5% that of thesham-treated controls (0.18 vs 34.0 μg insulin per pancreas, n=2). Thefact that these STZ-treated transgenic mice disposed of oral glucoselike normal mice despite having virtually no pancreatic β-cellsindicates that human insulin produced in the gut was sufficient tomaintain normal glucose tolerance.

These findings indicate that insulin production from gut K cells canprotect the mice from developing diabetes and also provide normalglucose homeostasis to the extent of restoring normal glucose tolerance.Therefore, insulin expressed in gut is a means with which hyperglycemicconditions such as diabetes can be treated.

Example IV

This example describes transplanting a transformed cell that produces aprotein in response to nutrient into a tissue of a mammalian subject.

To isolate target mucosal cells, a tissue biopsy will be collected fromthe duodenum of a subject. The biopsy is washed in ice-cold Hanks'balanced-salt solution (HBSS; Gibco BRL) ˜pH 7.4 containing 0.1% bovineserum albumin (BSA; Sigma) and finely chopped with scalpels followed bydigestion in an enzyme mixture containing 75 U/ml type I collagenase(Sigma), 75 U/ml type XI collagenase (Sigma), 0.9 U/ml type IXcollagenase (Sigma) and 1 U/ml trypsin (Worthington Biochemical Corp)for 1 hour in a shaking water bath at 37° C. The total volume is thendoubled with HBSS-BSA and allowed to settle for 10 min. The supernatantcontaining detached cells is discarded. The remaining tissue is furtherdigested in the enzyme mixture for two 45 min periods, with each stepfollowed by the addition of 300 μl of 0.5 M EDTA for 15 min. The cellsuspension resulting from digest 3 is filtered through Nitex mesh (200μm, B&SH Thompson) and washed and centrifuged at 200×g twice withHBSS-BSA supplemented with 0.01% dithiothreitol and 0.001% DNase. Thecells are then filtered a second time through fine Nitex mesh (62 μm,B&SH Thompson), counted, and diluted in HBSS-BSA-DTT-DNase to 6×10⁶cells/ml for elutriation.

Mucosal endocrine cells will be enriched using a counter-flowcentrifugal elutriation of cells (Lindahl P. E. Nature 161:648 (1948), aprocedure that separates cells on the basis of their sedimentationcoefficients. The cell suspension is pumped into a rotating chamber, andcells are held where their sedimentation rate is balanced by the flow offluid through the separation chamber. Different fractions of homogeneouscells are then ‘eluted’ by either increasing the flow rate through thechamber or decreasing the centrifugal speed. The appropriate flow ratesand centrifugation speeds are determined empirically. Batches consistingof 1.5×10⁸ dispersed cells are introduced into the Beckman elutriator(model J2-21 M/E; Beckman) via a pump (Cole Palmer) connected to asterile source of HBSS-BSA.

The enzyme dispersed mucosal cells are loaded into the elutriatorchamber at a rotor speed of 2500 rpm with a flow rate of 25 ml/min andwashed for 2 min. A 100 ml fraction (F1) is collected after changing theflow rate to 30 ml/min. A second 100 ml fraction (F2) is obtained at arotor speed of 2100 rpm and a flow rate of 55 ml/min. Cells from F2 areconcentrated by centrifuging at 200×g for 10 min, and then resuspendedin sterile culture medium (DMEM (47.5%) and Ham's F-12K containing 5.5mM glucose, 5% fetal calf serum, 2 ng/ml nerve growth factor, 8 mg/Linsulin, mg/L hydrocortisone, 50 mg/L gentamycin, 0.25 mg/L amphotericinB, 50 U/ml penicillin, 50 mg/L streptomycin and 20 μMcytosineβ-D-arabinofuranoside.

The resulting mucosal endocrine cells are conditionally immortalizedaccording to Kobayashi et. al. (Science 287:1258 (2000)). Culturedmucosal cells are transduced with a standard replication incompetentretroviral vector harboring a genetic construct consisting of the GIPpromoter operably-linked to an oncogene (e.g. telomerase, large-Tantigen, v-myc, ras, among others) tandemly fused to an IRES and aHSV-tk gene. The insulin and selection marker is expressedbi-cistronically. The genetic construct is flanked by recombinaserecognition sites to allow for excision of the oncogene and hencedeimmortalization of cells prior to transplantation.

To establish an immortalized K cell line, surviving clones ofcells—transduced with the retroviral vector carrying the GIP promoterlinked to an oncogene—are examined for the expression of GIP byimmuno-fluorescence staining and western blotting. Clones that expresssatisfactory amounts of GIP are further expanded to established a K cellline. The K cell line is further transduced with a retroviral vectorcarrying a genetic construct consisting of the GIP promoter operablylinked to a nucleic acid encoding human insulin and a positive selectionmarker. The insulin and selection marker is expressed bi-cistronically.The transfected K cells are incubated with appropriate selection drug.Surviving clones are isolated and tested for the expression of humaninsulin by western blot and ELISA (ALPCO).

K cell clone expressing appropriate levels of human insulin are cultureduntil sufficient number of cells are obtained. Prior to transplantationinto a mammalian subject, the human insulin expressing K cell line isdeimmortalized by excision of the oncogene. This is accomplished bytransfecting cells with adenovirus expressing the appropriaterecombinase (e.g. cre, flp, among others). Twenty-four to forty-eighthour after their transfection, cells are incubated in gancyclovir. After78 hrs exposure to gancyclovir, surviving cells (10⁶-10¹² cells) arepurified and prepared for transplantation into a mammalian subject.

As an alternative, mucosal precursor cells or stem cells are isolatedfrom duodenal biopsies by enzymatic (e.g. thermolysin) dissociation andexpanded in culture as described previously (Perraeault N. & Beaulieu J.F. Exp Cell Res 245:34 (1998); Perraeault N. & Beaulieu J. F. Exp CellRes 224:354 (1996)). These stem cells and precursor cells aretransfected with viral vectors carrying the GIP/Ins construct. Cellsthat are transfected successfully are selected by incubation inselection drug. These genetically engineered cells are then induced todifferentiate and finally transplanted into mammalian subjects.

In summary, this example illustrates an ex vivo method for engineering Kcells and mucosal endocrine precursor cells to produce human insulin.The engineered cells can be transplanted back into the same subject orto a different subject. The transplantation can be accomplished byseveral well established methodologies as disclosed herein or known inthe art. For example, cells can be encapsulated and implanted under theskin of the mammalian subject or cells can be implanted in the liverthrough portal delivery.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims

1.-70. (canceled)
 71. A method of treating diabetes, undesirable bodymass or obesity in a mammalian subject comprising contactinggastrointestinal mucosal tissue cells in the subject with apolynucleotide vector comprising a promoter in operable linkage with anucleic acid encoding GLP-1, wherein said contacting occurs in vivo viadelivery to stomach or small intestine, thereby producing transformedgastrointestinal mucosal tissue cells wherein orally feeding the subjectan amount of glucose, sucrose, fructose, carbohydrate, polypeptide,amino acid or fat increases transcription or secretion of the GLP-1 bythe transformed cells in an amount effective to treat diabetes,undesirable body mass or obesity in the mammalian subject.
 72. Themethod of claim 71, wherein the diabetes comprises type 1 diabetes. 73.The method of claim 71, wherein the subject has a fasting plasma glucoselevel greater than 110 mg/dl prior to treatment.
 74. The method of claim71, wherein the diabetes comprises insulin-independent (type 2)diabetes.
 75. The method of claim 71, wherein the glucose increasestranscription and secretion of the GLP-1 by the transformed cells. 76.The method of claim 71, wherein the polypeptide or amino acid increasessecretion of the GLP-1 by the transformed cells.
 77. The method of claim71, wherein the promoter comprises glucose-dependent insulinotropicpolypeptide (GIP) promoter or chromagranin A promoter.
 78. The method ofclaim 77, wherein the glucose-dependent insulinotropic polypeptide (GIP)or chromagranin A promoter comprises a functional variant or afunctional subsequence thereof, and wherein the glucose-dependentinsulinotropic polypeptide (GIP) or chromagranin A promoter functionalvariant or subsequence retains all or a part of non-variant orfull-length glucose-dependent insulinotropic polypeptide (GIP) orchromagranin A promoter transcription function.
 79. The method of claim71, wherein the gastrointestinal mucosal tissue cells are present in thesmall intestine.
 80. The method of claim 71, wherein the K cells, stemcells, multipotent progenitor cells or enteroendocrine cells are presentin the stomach.
 81. The method of claim 71, wherein said contactingproduces transformed K cells or transformed enteroendocrine cells. 82.The method of claim 71, wherein the vector comprises a viral vector. 83.The method of claim 71, wherein said contacting in vivo is with anendoscope, feeding tube, cannula, or catheter.
 84. The method of claim71, wherein said contacting in vivo occurs orally.
 85. The method ofclaim 71, wherein said vector comprises a vector that facilitatesintegration of the nucleic acid encoding insulin into the genome of saidK cells, stem cells, multipotent progenitor cells or enteroendocrinecells.
 86. The method of claim 71, wherein the subject is obese.
 87. Amethod of treating a blood coagulation disorder in a mammalian subjectcomprising contacting gastrointestinal mucosal cells in the subject witha polynucleotide vector comprising a promoter in operable linkage with anucleic acid encoding a clotting factor, wherein said contacting occursin vivo via delivery to stomach or small intestine, thereby producingtransformed gastrointestinal mucosal cells, wherein orally feeding thesubject an amount of glucose, sucrose, fructose, carbohydrate,polypeptide, amino acid or fat increases transcription or secretion ofthe clotting factor by the transformed cells in an amount effective totreat the blood coagulation disorder in the mammalian subject.